FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1994804526> ?p ?o ?g. }

• W1994804526 endingPage "28507" @default.
• W1994804526 startingPage "28501" @default.
• W1994804526 abstract "Closely opposed lesions form a unique class of DNA damage that is generated by ionizing radiation. Improper repair of closely opposed lesions could lead to the formation of double strand breaks that can result in increased lethality and mutagenesis. In vitro processing of closely opposed lesions was studied using double-stranded DNA containing a nick in close proximity opposite to a dihydrouracil. In this study we showed that HU protein, an Escherichia coli DNA-binding protein, has a role in the repair of closely opposed lesions. The repair of dihydrouracil is initiated by E. coli endonuclease III and processed via the base excision repair pathway. HU protein was shown to inhibit the rate of removal of dihydrouracil by endonuclease III only when the DNA substrate contained a nick in close proximity opposite to the dihydrouracil. In contrast, HU protein did not inhibit the subsequent steps of the base excision repair pathway, namely the DNA synthesis and ligation reactions catalyzed by E. coli DNA polymerase and E. coli DNA ligase, respectively. The nick-dependent selective inhibition of endonuclease III activity by HU protein suggests that HU could play a role in reducing the formation of double strand breaks in E. coli. Closely opposed lesions form a unique class of DNA damage that is generated by ionizing radiation. Improper repair of closely opposed lesions could lead to the formation of double strand breaks that can result in increased lethality and mutagenesis. In vitro processing of closely opposed lesions was studied using double-stranded DNA containing a nick in close proximity opposite to a dihydrouracil. In this study we showed that HU protein, an Escherichia coli DNA-binding protein, has a role in the repair of closely opposed lesions. The repair of dihydrouracil is initiated by E. coli endonuclease III and processed via the base excision repair pathway. HU protein was shown to inhibit the rate of removal of dihydrouracil by endonuclease III only when the DNA substrate contained a nick in close proximity opposite to the dihydrouracil. In contrast, HU protein did not inhibit the subsequent steps of the base excision repair pathway, namely the DNA synthesis and ligation reactions catalyzed by E. coli DNA polymerase and E. coli DNA ligase, respectively. The nick-dependent selective inhibition of endonuclease III activity by HU protein suggests that HU could play a role in reducing the formation of double strand breaks in E. coli. Ionizing radiation generates a wide spectrum of DNA damage, including strand breaks, abasic (AP) 1The abbreviations used are: AP, abasic; BER, base excision repair; DHU, dihydrouracil; Pol I, polymerase I.1The abbreviations used are: AP, abasic; BER, base excision repair; DHU, dihydrouracil; Pol I, polymerase I. sites, base damages, and cross-links (1von Sonntag C. The Chemical Basis of Radiation Biology. Taylor and Francis, London1987Google Scholar, 2Hall E.J. Radiobiology for the Radiobiologist. J. B. Lippincott Company, Philadelphia1994Google Scholar). The energy of ionizing radiation is not dispersed uniformly in the absorbing medium but as packets of energy along the tracks of the charge particles. Each deposition of energy can generate multiple ionization events that can lead to the generation of a unique class of lesion called clustered lesion (3Ward J.F. Progr. Nucl. Acids Mol. Biol. 1988; 35: 95-125Crossref PubMed Scopus (1170) Google Scholar, 4Sutherland B.M. Bennett P.V. Sidorkina O. Laval J. Biochemistry. 2000; 39: 8026-8031Crossref PubMed Scopus (190) Google Scholar, 5Sutherland B.M. Bennett P.V. Sidorkina O. Laval J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 103-108Crossref PubMed Scopus (403) Google Scholar, 6Chaudhry M.A. Weinfeld M. J. Mol. Biol. 1995; 249: 914-922Crossref PubMed Scopus (105) Google Scholar, 7Ward J.F. Radiat. Res. 1981; 86: 185-195Crossref PubMed Scopus (219) Google Scholar, 8Hall E.J. Radiology for the Radiologist. J. B. Lippincott Company, Philadelphia1993Google Scholar). In addition, when ionizing radiation interacts directly with DNA, multiple DNA ionizations can occur at the site of interaction that can also lead to the formation of clustered lesion. Thus, both the direct and indirect action of ionizing radiation on DNA can lead to the generation of clustered lesions (3Ward J.F. Progr. Nucl. Acids Mol. Biol. 1988; 35: 95-125Crossref PubMed Scopus (1170) Google Scholar, 4Sutherland B.M. Bennett P.V. Sidorkina O. Laval J. Biochemistry. 2000; 39: 8026-8031Crossref PubMed Scopus (190) Google Scholar, 5Sutherland B.M. Bennett P.V. Sidorkina O. Laval J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 103-108Crossref PubMed Scopus (403) Google Scholar, 6Chaudhry M.A. Weinfeld M. J. Mol. Biol. 1995; 249: 914-922Crossref PubMed Scopus (105) Google Scholar, 7Ward J.F. Radiat. Res. 1981; 86: 185-195Crossref PubMed Scopus (219) Google Scholar, 8Hall E.J. Radiology for the Radiologist. J. B. Lippincott Company, Philadelphia1993Google Scholar).The nature of clustered lesions is complex. They can consist of a combination of DNA breaks, AP sites, and base damages, either present on the same or opposing strands of DNA (4Sutherland B.M. Bennett P.V. Sidorkina O. Laval J. Biochemistry. 2000; 39: 8026-8031Crossref PubMed Scopus (190) Google Scholar, 5Sutherland B.M. Bennett P.V. Sidorkina O. Laval J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 103-108Crossref PubMed Scopus (403) Google Scholar, 6Chaudhry M.A. Weinfeld M. J. Mol. Biol. 1995; 249: 914-922Crossref PubMed Scopus (105) Google Scholar, 7Ward J.F. Radiat. Res. 1981; 86: 185-195Crossref PubMed Scopus (219) Google Scholar). A double strand break can be considered as a clustered lesion consisting of two single strand breaks located in close proximity on opposing strands of DNA. Much effort has been directed to understanding the repair of ionizing radiation-induced lesions such as strand breaks, AP sites, and base lesions; however, the biological process involved in the repair of these lesions within a clustered site is poorly understood. Strand breaks, AP sites, and base damages are repaired predominantly via the base excision repair (BER) pathway (9Freidberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society of Microbiology Press, Washington, D. C.1995Google Scholar, 10Wilson D.W. Engelward B.P. Samson L. Nickoloff J.A. Hoekstra M.F. DNA Repair in Prokaryotes and Lower Eukaryotes. Humana Press, Totowa, New Jersey1998: 29-64Google Scholar). When AP sites and base damages are processed via BER, intermediary strand breaks are generated. For clustered lesions consisting of DNA damage located on opposing strands of DNA, a double strand break can be generated if the excision of the second lesion is initiated before the first lesion is completely repaired. The formation of a double strand break can be more lethal and mutagenic than the original closely opposed lesions. To reduce the formation of double strand breaks during cellular processing of clustered lesions, it is likely that there are mechanisms within cells that help to reduce the probability of initiating the repair of a second lesion before the first lesion is fully repaired.It is interesting to note that many of the DNA repair glycosylases that initiate the repair of base lesions and AP sites are intrinsically inhibited by nearby nicks. Escherichia coli endonuclease III and formamidopyrimidine N-glycosylase activities are inhibited by nicks that are in close proximity and opposite to the base lesion (11Harrison L. Hatahet Z. Purmal A.A. Wallace S.S. Nucleic Acids Res. 1998; 26: 932-941Crossref PubMed Scopus (111) Google Scholar, 12David-Cordonnier M-H. Laval J. O'Neill P. J. Biol. Chem. 2000; 275: 11865-11873Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 13Takeshita M. Chang C.N. Johnson F. Will S. Grollman A.P. J. Biol. Chem. 1987; 262: 10171-10179Abstract Full Text PDF PubMed Google Scholar, 14Bourdat A.G. Gasparutto D. Cadet J. Nucleic Acids Res. 1999; 27: 1016-1024Crossref Scopus (69) Google Scholar). The observed inhibition could be due to the fact that a nick increases the flexibility of DNA around the remaining lesion. The increased flexibility could lead to a decrease in the affinity of these DNA glycosylases for lesions that are located close to a nick. However, prolonged incubation of DNA containing clustered damage with either endonuclease III or formamidopyrimidine N-glycosylase can still lead to the generation of double strand breaks. Because a nearby nick can only slow down the action of a repair glycosylase, it is therefore likely that additional mechanisms are necessary to further inhibit the action of DNA glycosylases so that generation of double strand breaks can be reduced during the repair of clustered lesions. We had show earlier that KU, a human double-stranded DNA-binding protein, can bind to a nick opposite dihydrouracil (DHU), leading to a significant inhibition of the human endonuclease III activity (15Hashimoto M. Donald C.D. Yannone S.M. Chen D.J. Roy R. Kow Y.W. J. Biol. Chem. 2001; 276: 12827-12831Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The increased inhibition of human endonuclease III activity by KU protein will help to reduce the possibility of generating double strand breaks during the repair of closely opposed lesions. Furthermore, even if a double strand break is formed, KU can bind to the ends of a newly generated double strand break and tether the two ends together as a protein-DNA complex, preventing the possibility of mis-joining (15Hashimoto M. Donald C.D. Yannone S.M. Chen D.J. Roy R. Kow Y.W. J. Biol. Chem. 2001; 276: 12827-12831Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar).In this study, we showed that the E. coli DNA-binding protein HU also inhibits the activity of E. coli endonuclease III when a nick is present in close proximity and opposite to a base damage. In contrast, HU protein did not inhibit significantly the subsequent steps of the repair process, namely DNA repair synthesis and DNA ligation. Thus HU could potentially mediate the sequential repair of a clustered lesion consisting of closely opposed DNA damage. These data further suggest that DNA-binding proteins that have high affinity for nicks, such as poly (ADP-ribose) DNA polymerase, could also mediate similar processes in the cells.EXPERIMENTAL PROCEDURESDNA Substrates—All oligonucleotides were obtained from Operon and purified by polyacrylamide gel (15%) electrophoresis as described previously (16Yao M. Kow Y.W. J. Biol. Chem. 1995; 270: 28609-28616Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Oligonucleotides were 5′-end-labeled with [γ-32P]ATP (Amersham Biosciences) using T4 polynucleotide kinase, following the instructions provided by the supplier. 32P-labeled oligonucleotides containing DHU were annealed to the appropriate complementary strands at 1:1.5 ratios in a buffer containing 10 mm Tris-HCl, pH 7.5, 0.1 m NaCl, and 2 mm mercaptoethanol by heating the mixture to 90 °C and cooling down slowly to room temperature. The oligonucleotide duplexes in Sequence I were used in this study (Q represents dihydrouracil). Duplex N contains a nick and a DHU. The nick is two nucleotides 3′ from DHU on the complementary strand. In contrast, duplex L contains only a single DHU.To determine the effect of distances between the nick and DHU on the inhibition of endonuclease III activity by HU protein, the substrates were prepared as in Sequence II.Sequence IIView Large Image Figure ViewerDownload Hi-res image Download (PPT)Duplex DNA containing a 5′ flap was constructed by hybridizing a 5′ 32P-labeled 33-mer and an unlabeled 16-mer with the complementary 30-mer. This will generate a duplex containing a 5′-labeled flap of 19 nucleotides long deriving from the unhybridized 5′ sequences of the 33-mer. The sequence of the three oligonucleotides for preparing the flap DNA is as follows: 33-mer, 5′-ATGTGGAAAATCTCTAGCAGGCTGCAGGTCGAC; 16-mer, 5′-CAGCAACGCAAGCTTG; 30-mer, 5′-GTCGACCTGCAGCCCAAGCTTGCGTTGCTG; and Sequence III, where (Fp) = flap, 5′-ATGTGGAAAATCTCTAGCA.Sequence IIIView Large Image Figure ViewerDownload Hi-res image Download (PPT)Enzymes and Proteins—E. coli endonuclease III was purified from an overproducing E. coli strains employing MonoS, MonoQ and phenyl-Sepharose column as described previously (17Asahara H. Wistort P.M. Bank J.F. Bakerian R.H. Cunningham R.P. Biochemistry. 1989; 10: 4444-4449Crossref Scopus (212) Google Scholar). Endonuclease V was purifed from an E. coli overproducing strain employing MonoS, MonoQ, and phenyl-Sepharose (18Yao M. Kow Y.W. J. Biol. Chem. 1997; 272: 30774-30779Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). A small amount of HU protein was initially obtained as a gift from Dr. Roger McMaken (John Hopkins University), and was later purified from E. coli strains overproducing HUα and HUβ subunits following published procedures (19Pellegrini O. Oberb J. Pinson V. Rouviere-Yaniv J. Biochimie (Paris). 2000; 82: 1-13Crossref PubMed Scopus (13) Google Scholar). HU protein is an 18-kDa heterodimeric protein consist of HUα and HUβ subunits, each subunit has a molecular mass of 9 kDa (19Pellegrini O. Oberb J. Pinson V. Rouviere-Yaniv J. Biochimie (Paris). 2000; 82: 1-13Crossref PubMed Scopus (13) Google Scholar). Briefly, hupA and hupB genes were PCR amplified from E. coli genomic DNA. The hupA gene was PCR-amplified using primers 5′-CCCCCCCCATATGAACAAGACTCAACTGATTGATGTAATT and 5′-CCCCCCCCTCGAGCTTAACTGCGTCTTTCCAGTCCTTGCCA, which introduce NdeI and XhoI restriction sites near the ends of the PCR fragment. Similarly, the hupB gene was PCR-amplified using primers 5′-CCCCCCCCATATGAAATAAATCTCAATTGATCGACAAGATT and 5′-CCCCCCCCTCGAGGTTTACCGCGTCTTTCAGTGCTTTACCT. Each of the PCR products were restricted with NdeI and XhoI, and ligated into pET22b(+) that was previously restricted with NdeI and XhoI, using T4 DNA ligase (16 °C for 16 h) and electroporated into E. coli BL21(pLysS), producing ampicillin-resistant colonies. The constructs thus generated (pHuA and pHuB) code for HUα and HUβ modified to contain six C-terminal histidines. BL21(pLysS) cells harboring either pHuA or pHuB were grown to 0.7 OD, and overproduction of HUα and Huβ were achieved by the addition of 0.5 mm of isopropyl-1-thio-β-d-galactopyranoside and continued to grow the cells for an additional 16 h at room temperature. HUα and Huβ proteins were then purified individually using nickel-nitrilotriacetic acid columns, following instructions supplied by the manufacturer (Novagen). E. coli DNA polymerase I and E. coli DNA ligase were purchased from USB biochemicals.Enzyme Assays—Endonuclease III activity was assayed in a standard reaction mix (10 μl) containing 0.1 m KCl, 10 mm Tris-HCl, pH 7.5, 50 fmol of labeled DNA substrates, and 20 fmol of endonuclease III. The reaction was performed at 37 °C for 10 min.Nick translation catalyzed by E. coli DNA polymerase I was performed in a buffer solution (10 μl) containing 67 mm potassium phosphate, pH 8.0, 6.7 mm MgCl2, 1 mm EDTA, 50 mm NaCl, 33 μm dATP, 33 μm dCTP, 33 μm dGTP, and 33 μm dTTP. Duplex N (see Sequence I) is composed of a 56-mer containing a DHU hybridized to two complementary DNA, a 21-mer and a 35-mer. The 35-mer was 5′-end-labeled with 32P and used as the primer for E. coli polymerase I-dependent nick translation DNA synthesis. The rate of formation of a labeled full-length product was used as an estimate for the E. coli polymerase I activity.DNA ligation was also performed with duplex N in a ligation buffer (10 μl) containing 30 mm Tris, pH 8.0, 4 mm MgCl2, 50 mm NaCl, 1 mm dithiothreitol, 20 μm NAD+, and 5 μg of bovine serum albumin. In this case, the 21-mer was 5′-end-labeled to provide the 5′-phosphoryl group that is required for the ligation reaction. The rate of formation of a full-length, 5′-end-labeled 56-mer was used for estimation for the DNA ligase activity.Endonuclease III, nick translation and DNA ligation reactions were stopped with 5 μl of a stop buffer containing 90% formamide, 10 mm EDTA, 0,.1% xylene, and 0.1% bromphenol blue. After the addition of a stop buffer, the reaction mixture was immediately heated at 90 °C for 10 min. A 5-μl sample was loaded onto a 12.5% denaturing polyacrylamide gel and electrophoresed at 2000 V for 1.5 h. The polyacrylamide gel was then dried and the amount of products formed was estimated by using the STORM PhosphoImager (Amersham Biosciences).RESULTSEffect of HU Protein on Endonuclease III Activity—HU protein binds tightly to various DNA replication and recombination intermediate structures (20Kamashev D. Rouviere-Yaniv J. EMBO J. 2000; 19: 6527-6535Crossref PubMed Scopus (143) Google Scholar, 21Kamashev D. Balandina A. Rouviere-Yaniv J. EMBO J. 1999; 18: 5434-5444Crossref PubMed Scopus (80) Google Scholar, 22Bonnefoy E. Takahashi M. Rouviere-Yaniv J. J. Mol. Biol. 1994; 242: 116-129Crossref PubMed Scopus (123) Google Scholar). In addition, it has a high affinity for DNA containing nicks and small gaps. Castaing et al. (23Castaing B. Zelwer C. Laval J. Boiteux S. J. Biol. Chem. 1995; 270: 10291-10296Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) showed that HU bound to DNA containing one base gap generated by DNA glycosylases. These data suggest that HU might play a role in preventing the untimely exonuclease digestion of DNA in cells by inhibiting the binding of exonuclease III to nicks and small gaps. Kamashev and Rouviere-Yaniv (20Kamashev D. Rouviere-Yaniv J. EMBO J. 2000; 19: 6527-6535Crossref PubMed Scopus (143) Google Scholar) showed that inhibition of exonuclease III activity by HU protein was only observed with DNA containing nicks. However, HU protein did not protect intact linear DNA from exonuclease III digestion, even at very high HU protein concentrations (20Kamashev D. Rouviere-Yaniv J. EMBO J. 2000; 19: 6527-6535Crossref PubMed Scopus (143) Google Scholar). Earlier, we showed that the human KU70/80 complex inhibited the rate of removal of DHU by human endonuclease III (15Hashimoto M. Donald C.D. Yannone S.M. Chen D.J. Roy R. Kow Y.W. J. Biol. Chem. 2001; 276: 12827-12831Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Inhibition was only observed when the DNA substrate contained a DHU that was close to and opposite a nick. Because HU protein also binds tightly to DNA containing a nick, it is thus expected that, like human KU protein, HU protein might also inhibit the endonuclease III activity. Fig. 1 shows the effect of increasing HU protein concentrations on the endonuclease III activity. Two DNA substrates were used in this experiment: duplex N, which contained a nick opposite and in close proximity to a DHU, and duplex L, which contains only a DHU. When duplex L was used as a substrate, increasing the amount of HU protein had no effect on the activity of E. coli endonuclease III (Fig. 1, panel A). However, when duplex N was used as the substrate, increasing the amount of HU protein led to a significant inhibition of the endonuclease III activity (Fig. 1, panel B). At 400 nm of HU protein, greater than 75% inhibition of endonuclease III activity was observed. This is comparable with the inhibition of E. coli exonuclease III activity by HU; at 400 nm HU inhibited ∼50% of the exonuclease activity of exonuclease III on nicked plasmid DNA (20Kamashev D. Rouviere-Yaniv J. EMBO J. 2000; 19: 6527-6535Crossref PubMed Scopus (143) Google Scholar).Fig. 1Effect of HU on endonuclease III activity. Panel A, 20 fmols of duplex L were incubated with 10 fmols of E. coli endonuclease III and an increasing amount of HU in a standard endonuclease III buffer at 37 °C for 10 min. The amount of endonuclease III-induced nicks was measured by electrophoresis on a 12.5% denaturing polyacrylamide gel. Duplex L incubated with HU only (♦) or with endonuclease III and HU (□). Panel B, 20 fmols of duplex N were incubated with 10 fmols of E. coli endonuclease III and an increasing amount of HU in a standard endonuclease III buffer at 37 °C for 10 min. Duplex N was incubated with HU only (♦) or with endonuclease III and HU (□).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine the effect that distances between the nick and DHU have on the inhibitory effect of HU on endonuclease III activity, five additional substrates were prepared with distances between the nick and DHU varied from 2, 4, 6, and 8 nucleotides lengths. Fig. 2 showed that HU exerted its maximum inhibitory effect when the nick was 2 nucleotides from the base lesion DHU. At 400 nm of HU protein, 50% of endonuclease III activity was inhibited when the nick was 2 nucleotides away, either 3′ or 5′ from DHU. When the nick was 4, 6, and 8 nucleotides away from DHU, inhibition of endonuclease III activity by HU was observed to be about 35, 22, and 12%, respectively.Fig. 2Effect of distance between the nick and DHU on the inhibitory effect of HU on endonuclease III activity. 20 fmols of duplex DNA containing nicks that are 0, 2, 4, 6, and 8 nucleotides from DHU on the complementary strand were incubated with 10 fmols of E. coli endonuclease III and 400 nm of HU in a standard endonuclease III buffer at 37 °C for 10 min. The amount of endonuclease III-induced nicks was measured by electrophoresis on a 12.5% denaturing polyacrylamide gel. Under the reaction condition used, endonuclease III activity on control DNA (CON) was 14.8 fmols of nick per 10 min (100%).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of HU on DNA Polymerase I Activity—To have sequential repair of closely opposed lesions, it is necessary that repair synthesis catalyzed by DNA polymerase I (Pol I) be carried out unimpeded on the DNA strand containing the newly generated nick, whereas the cleavage activity of endonuclease III on the opposing lesion is inhibited by HU protein. It is therefore expected that concentrations of HU protein that inhibit endonuclease III activity will have little effect on the activity of Pol I. The effect of increasing concentrations of HU protein on Pol I activity was examined by using duplex N with the 35-mer 5′-end-labeled with 32P. DNA synthesis, or DNA polymerase activity was measured by the rate of extension of the 5′-end-labeled 35-mer to the full-length 56-mer. Because E. coli Pol I contains both 3′-5′ and 5′-3′ exonuclease activity, the amount of Pol I used for this experiment was chosen by determining the amount of Pol I that will give a good rate of DNA synthesis but little degradation of labeled primer. Fig. 3 shows a titration of the amount of Pol I used in the competing reactions of primer extension and degradation. It was found that under the reaction conditions used, 0.01–0.05 units of Pol I gave an optimum rate of primer extension with little degradation of the primer (Fig. 3, lanes 6–8). However, higher concentrations of Pol I led to substantial degradation of the labeled 35-mer (Fig. 3, lanes 2–5). Based on these data, the effect of HU protein on the nick translation was studied with 0.01 units of DNA Pol I. Fig. 4 shows that increasing concentrations of HU protein (up to 400 nm) had little inhibitory effect on the Pol I nick translation-DNA synthesis activity (Fig. 4, panel A, lanes 5–8). Using 0.01 units of Pol I, a 10 min reaction converted 45% of the substrate to the expected 56-mer full-length product (Fig. 4, panel B). The extent of nick translation catalyzed by Pol I was little affected, even in the presence of 400 nm of HU protein (Fig. 4, panel B). Recent studies showed that in the presence of poly (ADP-ribose) DNA polymerase, the human polymerase β favors a strand displacement synthesis (24Prasad R. Lavrik O.I. Kim S.J. Kedar P. Yang X.P. Vande Berg B.J. Wilson S.H. J. Biol. Chem. 2001; 276: 2411-2414Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Because Pol I can also carry out strand displacement DNA synthesis, it is thus of interest to find out whether, in the presence of protein, Pol I can shift from nick translation to strand displacement DNA synthesis. To do this, both the 35-mer and the 21-mer of duplex N were 5′-end-labeled with 32P. Fig. 5 shows the time course of the reaction with Pol I. At an early time interval (1 min after the addition of Pol I), 400 nm of HU showed a slight inhibition on the rate of formation of the full-length 56-mer (panels A and B, lane 1). In the presence of 400 nm HU, the 1 min reaction generated predominantly primer extension products that are shorter than the full-length product (panel B, lane 1). However, as the reaction progressed, most of the extended products were extended to become full-length 56-mer (panel B, lanes 2–9). However, the rate of degradation of the 5′ DNA strand (the labeled 21-mer) was not inhibited (the faster migrating species of all lanes of panels A and B). These data suggest that high concentration of HU only slightly slowed down the initial extension of the 35-mer and appeared to have little effect on the progression of the nick translation reaction. This was indicated by the rapid formation of the full-length product and the extent of the degradation of the labeled 21-mer. These data thus suggest that the presence of HU protein did not shift Pol I from the nick translation mode of DNA synthesis to strand displacement mode of DNA repair synthesis.Fig. 3Titration of E. coli DNA polymerase I activity. 20 fmols of duplex N containing a 5′-labeled 35-mer was incubated in a standard nick translation buffer with decreasing E. coli DNA Pol I concentration. The nick translation reaction was performed at 37 °C for 10 min. At the end of 10 min, the reaction mix was assayed with a denaturing 12.5% polyacrylamide gel for the formation of full-length nick translation product. Lane 1, control duplex N showing the 5′-labeled 35-mer (primer). Lane 2–8, duplex N was incubated with 1 unit (lane 2), 0.5 units (lane 3), 0.25 units (lane 4), 0.1 units (lane 5), 0.05 units (lane 6), 0.025 units (lane 7), and 0.01 units (lane 8) of Pol I.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Effect of HU on DNA polymerase I activity. Panel A, DNA polymerase I activity was measured by its nick translation activity. 20 fmols of duplex N containing a 5′-labeled 35-mer was incubated in a standard nick translation buffer with 0.01 unit of E. coli DNA Pol I and an increasing amount of HU protein. The nick translation reaction was performed at 37 °C for 10 min. At the end of 10 min, the reaction mix was assayed with a denaturing 12.5% polyacrylamide gel for the formation of full-length nick translation product. Lanes 1–4, duplex N was incubated with 0 nm (lane 1), 100 nm (lane 2), 200 nm (lane 3), and 400 nm (lane 4) of HU protein. Lanes 5–8, duplex N was incubated with 0.01 unit of Pol I and 0 nm (lane 5), 100 nm (lane 6), 200 nm (lane 7), and 400 nm (lane 8) of HU protein. Panel B, nick translation assays performed in panel A were quantified using the Storm PhsophoImager (Amersham Biosciences). Duplex N was incubated with increasing amounts of HU (□) or with 0.01 unit of Pol I and increasing amounts of HU (▵).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Effect of HU on the 5′-3′ exonuclease activity of DNA polymerase I. 20 fmols of duplex N containing a 5′-labeled 35-mer and 5′-end-labeled 21-mer was incubated in a standard nick translation buffer with 0.01 unit of E. coli DNA Pol I and 400 nm of HU protein. The nick translation was assayed by the formation of a full-length 56-mer and the 5′-3 exonuclease activity by the formation of labeled nucleotide shorter than the 21-mer. Lane C, control duplex N containing the 5′-labeled 35-mer and 21-mer. Panel A, nick translation was performed in the absence of HU protein. Panel B, nick translation was performed in the presence of 400 nm HU protein. For both panels A and B, 1 min reaction (lane 1), 2 min reaction (lane 2), 3 min reaction (lane 3), 4 min reaction (lane 4), 5 min reaction (lane 5), 8 min reaction (lane 6), 10 min reaction (lane 7), 12 min reaction (lane 8), and 15 min reaction (lane 9).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In addition to being a DNA polymerase, Pol I is also a flap endonuclease that is capable of cleaving the 5′ flap from a flap DNA structure (25Lyamichev V.D. Brow M.A. Dahlberg J.E. Science. 1993; 260: 778-783Crossref PubMed Scopus (301) Google Scholar). Flap DNA structures are thought to be formed on the DNA lagging strand during processing of Okazaki fragment (26MacNeill S.A. Curr. Biol. 2001; 11: R842-R844Abstract Full Text Full Text PDF PubMed Scopus (48) Google Schola" @default.
• W1994804526 created "2016-06-24" @default.
• W1994804526 creator A5000385147 @default.
• W1994804526 creator A5004831596 @default.
• W1994804526 creator A5008081357 @default.
• W1994804526 creator A5058799419 @default.
• W1994804526 date "2003-08-01" @default.
• W1994804526 modified "2023-09-30" @default.
• W1994804526 title "HU Protein of Escherichia coli Has a Role in the Repair of Closely Opposed Lesions in DNA" @default.
• W1994804526 cites W1577909016 @default.
• W1994804526 cites W1602417853 @default.
• W1994804526 cites W1917341586 @default.
• W1994804526 cites W1965218097 @default.
• W1994804526 cites W1965573008 @default.
• W1994804526 cites W1967634425 @default.
• W1994804526 cites W1982305908 @default.
• W1994804526 cites W1982321678 @default.
• W1994804526 cites W1982461902 @default.
• W1994804526 cites W1989252766 @default.
• W1994804526 cites W1990491149 @default.
• W1994804526 cites W1990942802 @default.
• W1994804526 cites W1996281999 @default.
• W1994804526 cites W1999325571 @default.
• W1994804526 cites W2005318857 @default.
• W1994804526 cites W2006016527 @default.
• W1994804526 cites W2011320725 @default.
• W1994804526 cites W2020641167 @default.
• W1994804526 cites W2024060947 @default.
• W1994804526 cites W2025459414 @default.
• W1994804526 cites W2028067449 @default.
• W1994804526 cites W2033270381 @default.
• W1994804526 cites W2034129646 @default.
• W1994804526 cites W2043252165 @default.
• W1994804526 cites W2054373709 @default.
• W1994804526 cites W2085083586 @default.
• W1994804526 cites W2093181144 @default.
• W1994804526 cites W2111313313 @default.
• W1994804526 cites W2113375881 @default.
• W1994804526 cites W2117261809 @default.
• W1994804526 cites W2163076785 @default.
• W1994804526 cites W2170482532 @default.
• W1994804526 doi "https://doi.org/10.1074/jbc.m303970200" @default.
• W1994804526 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12748168" @default.
• W1994804526 hasPublicationYear "2003" @default.
• W1994804526 type Work @default.
• W1994804526 sameAs 1994804526 @default.
• W1994804526 citedByCount "26" @default.
• W1994804526 countsByYear W19948045262013 @default.
• W1994804526 countsByYear W19948045262014 @default.
• W1994804526 countsByYear W19948045262015 @default.
• W1994804526 countsByYear W19948045262016 @default.
• W1994804526 countsByYear W19948045262017 @default.
• W1994804526 countsByYear W19948045262019 @default.
• W1994804526 countsByYear W19948045262022 @default.
• W1994804526 crossrefType "journal-article" @default.
• W1994804526 hasAuthorship W1994804526A5000385147 @default.
• W1994804526 hasAuthorship W1994804526A5004831596 @default.
• W1994804526 hasAuthorship W1994804526A5008081357 @default.
• W1994804526 hasAuthorship W1994804526A5058799419 @default.
• W1994804526 hasBestOaLocation W19948045261 @default.
• W1994804526 hasConcept C104317684 @default.
• W1994804526 hasConcept C134935766 @default.
• W1994804526 hasConcept C54355233 @default.
• W1994804526 hasConcept C547475151 @default.
• W1994804526 hasConcept C552990157 @default.
• W1994804526 hasConcept C70721500 @default.
• W1994804526 hasConcept C86803240 @default.
• W1994804526 hasConcept C89423630 @default.
• W1994804526 hasConceptScore W1994804526C104317684 @default.
• W1994804526 hasConceptScore W1994804526C134935766 @default.
• W1994804526 hasConceptScore W1994804526C54355233 @default.
• W1994804526 hasConceptScore W1994804526C547475151 @default.
• W1994804526 hasConceptScore W1994804526C552990157 @default.
• W1994804526 hasConceptScore W1994804526C70721500 @default.
• W1994804526 hasConceptScore W1994804526C86803240 @default.
• W1994804526 hasConceptScore W1994804526C89423630 @default.
• W1994804526 hasIssue "31" @default.
• W1994804526 hasLocation W19948045261 @default.
• W1994804526 hasOpenAccess W1994804526 @default.
• W1994804526 hasPrimaryLocation W19948045261 @default.
• W1994804526 hasRelatedWork W1991523530 @default.
• W1994804526 hasRelatedWork W2002128513 @default.
• W1994804526 hasRelatedWork W2020824267 @default.
• W1994804526 hasRelatedWork W2057739827 @default.
• W1994804526 hasRelatedWork W2075354549 @default.
• W1994804526 hasRelatedWork W2080910126 @default.
• W1994804526 hasRelatedWork W2342146826 @default.
• W1994804526 hasRelatedWork W2359817556 @default.
• W1994804526 hasRelatedWork W2414680297 @default.
• W1994804526 hasRelatedWork W2092874662 @default.
• W1994804526 hasVolume "278" @default.
• W1994804526 isParatext "false" @default.
• W1994804526 isRetracted "false" @default.
• W1994804526 magId "1994804526" @default.
• W1994804526 workType "article" @default.