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1.
XPA is a central protein component of nucleotide excision repair (NER), a ubiquitous, multi-component cellular pathway responsible for the removal and repair of many structurally distinct DNA lesions from the eukaryotic genome. The solution structure of the minimal DNA-binding domain of XPA (XPA-MBD: M98-F219) has recently been determined and chemical shift mapping experiments with 15N-labeled XPA-MBD show that XPA binds DNA along a basic surface located in the C-terminal loop-rich subdomain. Here, XPA-DNA interactions are further characterized using an XPA fragment containing the minimal DNA-binding domain plus the ERCC1-binding region (XPA-EM: M59-F219). The 15N/1H HSQC spectrum of XPA-EM closely maps onto the 15N/1H HSQC spectrum of XPA-MBD, suggesting the DNA-binding domain is intact in the larger XPA fragment. Such a conclusion is corroborated by chemical shift mapping experiments of XPA-EM with a single strand DNA oligomer, dCCAATAACC (d9), that show the same set of 15N/1H HSQC cross peaks are effected by the addition of DNA. However, relative to DNA-free XPA-MBD, the 15N/1H HSQC cross peaks of many of the basic residues in the loop-rich subdomain of DNA-free XPA-EM are less intense, or gone altogether, suggesting the acidic ERRC1-binding region of XPA-EM may associate transiently with the basic DNA-binding surface. While the DNA-binding domain in XPA-EM is structured and functional, 15N-edited NOESY spectra of XPA-EM indicate that the acidic ERRC1-binding region is unstructured. If the structural features observed for XPA-EM persist in XPA, transient intramolecular association of the ERCC1-binding domain with the DNA-binding region may play a role in the sequential assembly of the NER components. 相似文献
2.
Daughdrill GW Buchko GW Botuyan MV Arrowsmith C Wold MS Kennedy MA Lowry DF 《Nucleic acids research》2003,31(14):4176-4183
Replication protein A (RPA) is a heterotrimeric single-stranded DNA- (ssDNA) binding protein that can form a complex with the xeroderma pigmentosum group A protein (XPA). This complex can preferentially recognize UV-damaged DNA over undamaged DNA and has been implicated in the stabilization of open complex formation during nucleotide excision repair. In this report, nuclear magnetic resonance (NMR) spectroscopy was used to investigate the interaction between a fragment of the 70 kDa subunit of human RPA, residues 1–326 (hRPA701–326), and a fragment of the human XPA protein, residues 98–219 (XPA-MBD). Intensity changes were observed for amide resonances in the 1H–15N correlation spectrum of uniformly 15N-labeled hRPA701–326 after the addition of unlabeled XPA-MBD. The intensity changes observed were restricted to an ssDNA-binding domain that is between residues 183 and 296 of the hRPA701–326 fragment. The hRPA701–326 residues with the largest resonance intensity reductions were mapped onto the structure of the ssDNA-binding domain to identify the binding surface with XPA-MBD. The XPA-MBD-binding surface showed significant overlap with an ssDNA-binding surface that was previously identified using NMR spectroscopy and X-ray crystallography. Overlapping XPA-MBD- and ssDNA-binding sites on hRPA701–326 suggests that a competitive binding mechanism mediates the formation of the RPA–XPA complex. To determine whether a ternary complex could form between hRPA701–326, XPA-MBD and ssDNA, a 1H–15N correlation spectrum was acquired for uniformly 15N-labeled hRPA701–326 after the simultaneous addition of unlabeled XPA-MBD and ssDNA. In this experiment, the same chemical shift perturbations were observed for hRPA701–326 in the presence of XPA-MBD and ssDNA as was previously observed in the presence of ssDNA alone. The ability of ssDNA to compete with XPA-MBD for an overlapping binding site on hRPA701–326 suggests that any complex formation between RPA and XPA that involves the interaction between XPA-MBD and hRPA701–326 may be modulated by ssDNA. 相似文献
3.
Buchko GW Daughdrill GW de Lorimier R Rao B K Isern NG Lingbeck JM Taylor JS Wold MS Gochin M Spicer LD Lowry DF Kennedy MA 《Biochemistry》1999,38(46):15116-15128
Human XPA is an essential component in the multienzyme nucleotide excision repair (NER) pathway. The solution structure of the minimal DNA binding domain of XPA (XPA-MBD: M98-F219) was recently determined [Buchko et al. (1998) Nucleic Acids Res. 26, 2779-2788, Ikegami et al. (1998) Nat. Struct. Biol. 5, 701-706] and shown to consist of a compact zinc-binding core and a loop-rich C-terminal subdomain connected by a linker sequence. Here, the solution structure of XPA-MBD was further refined using an entirely new class of restraints based on pseudocontact shifts measured in cobalt-substituted XPA-MBD. Using this structure, the surface of XPA-MBD which interacts with DNA and a fragment of the largest subunit of replication protein A (RPA70 Delta C327: M1-Y326) was determined using chemical shift mapping. DNA binding in XPA-MBD was highly localized in the loop-rich subdomain for DNA with or without a lesion [dihydrothymidine (dhT) or 6-4-thymidine-cytidine (64TC)], or with DNA in single- or double-stranded form, indicating that the character of the lesion itself is not the driving force for XPA binding DNA. RPA70 Delta C327 was found to contact regions in both the zinc-binding and loop-rich subdomains. Some overlap of the DNA and RPA70 Delta C327 binding regions was observed in the loop-rich subdomain, indicating a possible cooperative DNA-binding mode between XPA and RPA70 Delta C327. To complement the chemical shift mapping data, the backbone dynamics of free XPA-MBD and XPA-MBD bound to DNA oligomers containing dhT or 64TC lesions were investigated using 15N NMR relaxation data. The dynamic analyses for the XPA-MBD complexes with DNA revealed localized increases and decreases in S2 and an increase in the global correlation time. Regions of XPA-MBD with the largest increases in S2 overlapped regions having the largest chemical shifts changes upon binding DNA, indicating that the loop-rich subdomain becomes more rigid upon binding DNA. Interestingly, S2 decreased for some residues in the zinc-binding core upon DNA association, indicating a possible concerted structural rearrangement on binding DNA. 相似文献
4.
Human nucleotide excision repair protein XPA: extended X-ray absorption fine-structure evidence for a metal-binding domain. 总被引:3,自引:0,他引:3
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N. J. Hess G. W. Buchko S. D. Conradson F. J. Espinosa S. Ni B. D. Thrall M. A. Kennedy 《Protein science : a publication of the Protein Society》1998,7(9):1970-1975
The ubiquitous, multi-enzyme, nucleotide excision repair (NER) pathway is responsible for correcting a wide range of chemically and structurally distinct DNA lesions in the eukaryotic genome. Human XPA, a 31 kDa, zinc-associated protein, is thought to play a major NER role in the recognition of damaged DNA and the recruitment of other proteins, including RPA, ERCC1, and TFIIH, to repair the damage. Sequence analyses and genetic evidence suggest that zinc is associated with a C4-type motif, C105-X2-C108-X17-C126-X2-C129, located in the minimal DNA binding region of XPA (M98-F219). The zinc-associated motif is essential for damaged DNA recognition. Extended X-ray absorption fine structure (EXAFS) spectra collected on the zinc associated minimal DNA-binding domain of XPA (ZnXPA-MBD) show directly, for the first time, that the zinc is coordinated to the sulfur atoms of four cysteine residues with an average Zn-S bond length of 2.34+/-0.01 A. XPA-MBD was also expressed in minimal medium supplemented with cobalt nitrate to yield a blue-colored protein that was primarily (>95%) cobalt associated (CoXPA-MBD). EXAFS spectra collected on CoXPA-MBD show that the cobalt is also coordinated to the sulfur atoms of four cysteine residues with an average Co-S bond length of 2.33+/-0.02 A. 相似文献
5.
NMR study on the interaction between RPA and DNA decamer containing cis-syn cyclobutane pyrimidine dimer in the presence of XPA: implication for damage verification and strand-specific dual incision in nucleotide excision repair 总被引:1,自引:0,他引:1
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In mammalian cells, nucleotide excision repair (NER) is the major pathway for the removal of bulky DNA adducts. Many of the key NER proteins are members of the XP family (XPA, XPB, etc.), which was named on the basis of its association with the disorder xerodoma pigmentosum. Human replication protein A (RPA), the ubiquitous single-stranded DNA-binding protein, is another of the essential proteins for NER. RPA stimulates the interaction of XPA with damaged DNA by forming an RPA–XPA complex on damaged DNA sites. Binding of RPA to the undamaged DNA strand is most important during NER, because XPA, which directs the excision nucleases XPG and XPF, must bind to the damaged strand. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to assess the binding of the tandem high affinity DNA-binding domains, RPA-AB, and of the isolated domain RPA-A, to normal DNA and damaged DNA containing the cyclobutane pyrimidine dimer (CPD) lesion. Both RPA-A and RPA-AB were found to bind non- specifically to both strands of normal and CPD- containing DNA duplexes. There were no differences observed when binding to normal DNA duplex was examined in the presence of the minimal DNA-binding domain of XPA (XPA-MBD). However, there is a drastic difference for CPD-damaged DNA duplex as both RPA-A and RPA-AB bind specifically to the undamaged strand. The strand-specific binding of RPA and XPA to the damaged duplex DNA shows that RPA and XPA play crucial roles in damage verification and guiding cleavage of damaged DNA during NER. 相似文献
6.
Nucleotide excision repair (NER) is the process responsible for eliminating most ultraviolet (UV) radiation damage from DNA, as well as base alterations caused by a variety of mutagens. The xeroderma pigmentosum group A complementing protein (XPA) is believed to be involved in the early step of NER by recognizing and binding damaged DNA. Recent work has suggested that electrospray ionization-mass spectrometry (ESI-MS) can be an effective tool for the study of protein-DNA complexes. We have used ESI-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to examine the cisplatin-adducted oligonucleotide and its interaction with the human XPA minimal binding domain (XPA-MBD). High-resolution FTICR experiments of the binding products showed that both double-stranded damaged 20-mer and double-stranded undamaged 20-mer formed 1:1 noncovalent complexes with XPA-MBD. A 2:1 binding stoichiometry complex was also observed between XPA-MBD and double-stranded damaged 20-mer. Competitive binding experiments indicated only slightly preferential binding of XPA-MBD with the double-stranded damaged 20-mer compared to the undamaged 20-mer. The results demonstrate that ESI-FTICR mass spectrometry provides a fast and efficient approach for characterizing weak protein-DNA interactions such as the binding between XPA-MBD and a 20-mer oligonucleotide system. 相似文献
7.
Yuliya S. Krasikova Nadejda I. Rechkunova Ekaterina A. Maltseva Irina O. Petruseva Olga I. Lavrik 《Nucleic acids research》2010,38(22):8083-8094
The interaction of xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA) with damaged DNA in nucleotide excision repair (NER) was studied using model dsDNA and bubble-DNA structure with 5-{3-[6-(carboxyamido-fluoresceinyl)amidocapromoyl]allyl}-dUMP lesions in one strand and containing photoreactive 5-iodo-dUMP residues in defined positions. Interactions of XPA and RPA with damaged and undamaged DNA strands were investigated by DNA–protein photocrosslinking and gel shift analysis. XPA showed two maximums of crosslinking intensities located on the 5′-side from a lesion. RPA mainly localized on undamaged strand of damaged DNA duplex and damaged bubble-DNA structure. These results presented for the first time the direct evidence for the localization of XPA in the 5′-side of the lesion and suggested the key role of XPA orientation in conjunction with RPA binding to undamaged strand for the positioning of the NER preincision complex. The findings supported the mechanism of loading of the heterodimer consisting of excision repair cross-complementing group 1 and xeroderma pigmentosum group F proteins by XPA on the 5′-side from the lesion before damaged strand incision. Importantly, the proper orientation of XPA and RPA in the stage of preincision was achieved in the absence of TFIIH and XPG. 相似文献
8.
The TREX enzymes process DNA as the major 3′→5′ exonuclease activity in mammalian cells. TREX2 and TREX1 are members of the DnaQ family of exonucleases and utilize a two metal ion catalytic mechanism of hydrolysis. The structure of the dimeric TREX2 enzyme in complex with single-stranded DNA has revealed binding properties that are distinct from the TREX1 protein. The TREX2 protein undergoes a conformational change in the active site upon DNA binding including ordering of active site residues and a shift of an active site helix. Surprisingly, even when a single monomer binds DNA, both monomers in the dimer undergo the structural rearrangement. From this we have proposed a model for DNA binding and 3′ hydrolysis for the TREX2 dimer. The structure also shows how TREX proteins potentially interact with double-stranded DNA and suggest features that might be involved in strand denaturation to provide a single-stranded substrate for the active site. 相似文献
9.
Proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication, DNA repair, cell cycle regulation, chromatin remodeling, and epigenetics. Many proteins interact with PCNA through the PCNA interacting peptide (PIP)-box or the newly identified AlkB homolog 2 PCNA interacting motif (APIM). The xeroderma pigmentosum group A (XPA) protein, with a central but somewhat elusive role in nucleotide excision repair (NER), contains the APIM sequence suggesting an interaction with PCNA. With an in vivo based approach, using modern techniques in live human cells, we show that APIM in XPA is a functional PCNA interacting motif and that efficient NER of UV lesions is dependent on an intact APIM sequence in XPA. We show that XPA−/− cells complemented with XPA containing a mutated APIM sequence have increased UV sensitivity, reduced repair of cyclobutane pyrimidine dimers and (6–4) photoproducts, and are consequently more arrested in S phase as compared to XPA−/− cells complemented with wild type XPA. Notably, XPA colocalizes with PCNA in replication foci and is loaded on newly synthesized DNA in undamaged cells. In addition, the TFIIH subunit XPD, as well as XPF are loaded on DNA together with XPA, and XPC and XPG colocalize with PCNA in replication foci. Altogether, our results suggest a presence of the NER complex in the vicinity of the replisome and a novel role of NER in post-replicative repair. 相似文献
10.
Preferential binding of human XPA to the mitomycin C-DNA interstrand crosslink and modulation by arsenic and cadmium 总被引:1,自引:0,他引:1
The Xeroderma Pigmentosum A (XPA) protein is involved in the DNA damage recognition and repair complex formation steps of nucleotide excision repair (NER), and has been shown to preferentially bind to various forms of DNA damage including bulky lesions. DNA interstrand crosslinks are of particular interest as a form of DNA damage, since these lesions involve both strands of duplex DNA and present special challenges to the repair machinery, and mitomycin C (MMC) is one of several useful cancer chemotherapy drugs that induce these lesions. Purified XPA and the minimal DNA-binding domain of XPA are both fully capable of preferentially binding to MMC-DNA interstrand crosslinks in the absence of other proteins from the NER complex. Circular dichroism (CD) and gel shift assays were used to investigate XPA-DNA binding and to assess changes in secondary structure induced as a consequence of the interaction of XPA with model MMC-crosslinked and unmodified DNAs. These studies revealed that while XPA demonstrates only a modest increase in affinity for adducted DNA, it adopts a different conformation when bound to MMC-damaged DNA than when bound to undamaged DNA. This change in conformation may be more important in recruiting other proteins into a competent NER complex at damaged sites than preferential binding per se. Arsenic had little effect on XPA binding even at toxic concentrations, whereas cadmium reduced XPA binding to DNA to 10-15% that of Zn-XPA, and zinc addition could only partially restore activity. In addition, there was little or no change in conformation when Cd-XPA bound MMC-crosslinked DNA even though it demonstrated preferential binding, which may contribute to the mechanism by which cadmium can act as a co-mutagen and co-carcinogen. 相似文献
11.
The nature of the 5'-terminus is a major determinant for DNA processing by Schizosaccharomyces pombe Rad2p, a FEN-1 family nuclease
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The nuclease activity of FEN-1 is essential for both DNA replication and repair. Intermediate DNA products formed during these processes possess a variety of structures and termini. We have previously demonstrated that the 5′→3′ exonuclease activity of the Schizosaccharomyces pombe FEN-1 protein Rad2p requires a 5′-phosphoryl moiety to efficiently degrade a nick-containing substrate in a reconstituted alternative excision repair system. Here we report the effect of different 5′-terminal moieties of a variety of DNA substrates on Rad2p activity. We also show that Rad2p possesses a 5′→3′ single-stranded exonuclease activity, similar to Saccharomyces cerevisiae Rad27p and phage T5 5′→3′ exonuclease (also a FEN-1 homolog). FEN-1 nucleases have been associated with the base excision repair pathway, specifically processing cleaved abasic sites. Because several enzymes cleave abasic sites through different mechanisms resulting in different 5′-termini, we investigated the ability of Rad2p to process several different types of cleaved abasic sites. With varying efficiency, Rad2p degrades the products of an abasic site cleaved by Escherichia coli endonuclease III and endonuclease IV (prototype AP endonucleases) and S.pombe Uve1p. These results provide important insights into the roles of Rad2p in DNA repair processes in S.pombe. 相似文献
12.
Partial purification and characterization of four endodeoxyribonuclease activities from Escherichia coli K-12 总被引:1,自引:0,他引:1
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Four hitherto undescribed endodeoxyribonucleases, temporarily designated A1, A2, A3, and B, have been isolated from E. coli K-12. Each requires Mg++ and is not stimulated by ATP or S-adenosylmethionine. A3 is strongly inhibited by Fe+++ and weakly inhibited by ATP, S-adenosylmethionine, and DPN, whereas B is inhibited by caffeine. Each can be purified free of exonuclease or DNA-3′-phosphatase. A1 (molecular weight approximately 72,000) cleaves single-stranded, circular fd DNA to form 3′-hydroxyl termini and introduces nicks and breaks in the closed, double-stranded replicative form DNA of fd (fd RFI). A2 (molecular weight approximately 46,000) cleaves fd DNA and introduces nicks and breaks in RFI, forming 3′-hydroxyl- and 5′-phosphoryl termini. A3 (molecular weight approximately 38,000) cleaves fd DNA to form 3′-hydroxyl termini and introduces only nicks in fd RFI. Irradiation of the RFI with ultraviolet light markedly increases the rate of hydrolysis by A3. B appears to form 3′-phosphoryl termini with fd DNA, but its characterization is highly preliminary due to its instability. 相似文献
13.
The RAD9–RAD1–HUS1 (9–1–1) clamp forms one half of the DNA damage checkpoint system that signals the presence of substantial regions of single-stranded DNA arising from replication fork collapse or resection of DNA double strand breaks. Loaded at the 5′-recessed end of a dsDNA–ssDNA junction by the RAD17–RFC clamp loader complex, the phosphorylated C-terminal tail of the RAD9 subunit of 9–1–1 engages with the mediator scaffold TOPBP1 which in turn activates the ATR kinase, localised through the interaction of its constitutive partner ATRIP with RPA-coated ssDNA. Using cryogenic electron microscopy (cryoEM) we have determined the structure of a complex of the human RAD17–RFC clamp loader bound to human 9–1–1, engaged with a dsDNA–ssDNA junction. The structure answers the key questions of how RAD17 confers specificity for 9–1–1 over PCNA, and how the clamp loader specifically recognises the recessed 5′ DNA end and fixes the orientation of 9–1–1 on the ssDNA. 相似文献
14.
Mechanism of degradation of duplex DNA by the DNase induced by herpes simplex virus 总被引:7,自引:6,他引:1
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P J Hoffmann 《Journal of virology》1981,38(3):1005-1014
Reaction intermediates formed during the degradation of linear PM2, T5, and λ DNA by herpes simplex virus (HSV) DNase have been examined by agarose gel electrophoresis. Digestion of T5 DNA by HSV type 2 (HSV-2) DNase in the presence of Mn2+ (endonuclease only) gave rise to 6 major and 12 minor fragments. Some of the fragments produced correspond to those observed after cleavage of T5 DNA by the single-strand-specific S1 nuclease, indicating that the HSV DNase rapidly cleaves opposite a nick or gap in a duplex DNA molecule. In contrast, HSV DNase did not produce distinct fragments upon digestion of linear PM2 or λ DNA, which do not contain nicks. In the presence of Mg2+, when both endonuclease and exonuclease activities of the HSV DNase occur, most of the same distinct fragments from digestion of T5 DNA were observed. However, these fragments were then further degraded preferentially from the ends, presumably by the action of the exonuclease activity. Unit-length λ DNA, EcoRI restriction fragments of λ DNA, and linear PM2 DNA were also degraded from the ends by HSV DNase in the same manner. Previous studies have suggested that the HSV exonuclease degrades in the 3′ → 5′ direction. If this is correct, and since only 5′-monophosphate nucleosides are produced, then HSV DNase should “activate” DNA for DNA polymerase. However, unlike pancreatic DNase I, neither HSV-1 nor HSV-2 DNase, in the presence of Mg2+ or Mn2+, activated calf thymus DNA for HSV DNA polymerase. This suggests that HSV DNase degrades both strands of a linear double-stranded DNA molecule from the same end at about the same rate. That is, HSV DNase is apparently capable of degrading DNA strands in the 3′ → 5′ direction as well as in the 5′ → 3′ direction, yielding progressively smaller double-stranded molecules with flush ends. Except with minor differences, HSV-1 and HSV-2 DNases act in a similar manner. 相似文献
15.
Thomas J. Albert Jason Norton Markus Ott Todd Richmond Kate Nuwaysir Emile F. Nuwaysir Klaus-Peter Stengele Roland D. Green 《Nucleic acids research》2003,31(7):e35
Light-directed synthesis of high-density microarrays is currently performed in the 3′→5′ direction due to constraints in existing synthesis chemistry. This results in the probes being unavailable for many common types of enzymatic modification. Arrays that are synthesized in the 5′→3′ direction could be utilized to perform parallel genotyping and resequencing directly on the array surface, dramatically increasing the throughput and reducing the cost relative to existing techniques. In this report we demonstrate the use of photoprotected phosphoramidite monomers for light-directed array synthesis in the 5′→3′ direction, using maskless array synthesis technology. These arrays have a dynamic range of >2.5 orders of magnitude, sensitivity below 1 pM and a coefficient of variance of <10% across the array surface. Arrays containing >150 000 probe sequences were hybridized to labeled mouse cRNA producing highly concordant data (average R2 = 0.998). We have also shown that the 3′ ends of array probes are available for sequence-specific primer extension and ligation reactions. 相似文献
16.
Werner’s syndrome (WS) is an autosomal recessive disorder in humans characterized by the premature development of a partial array of age-associated pathologies. WRN, the gene defective in WS, encodes a 1432 amino acid protein (hWRN) with intrinsic 3′→5′ DNA helicase activity. We recently showed that hWRN is also a 3′→5′ exonuclease. Here, we further characterize the hWRN exonuclease. hWRN efficiently degraded the 3′ recessed strands of double-stranded DNA or a DNA–RNA heteroduplex. It had little or no activity on blunt-ended DNA, DNA with a 3′ protruding strand, or single-stranded DNA. The hWRN exonuclease efficiently removed a mismatched nucleotide at a 3′ recessed terminus, and was capable of initiating DNA degradation from a 12-nt gap, or a nick. We further show that the mouse WRN (mWRN) is also a 3′→5′ exonuclease, with substrate specificity similar to that of hWRN. Finally, we show that hWRN forms a trimer and interacts with the proliferating cell nuclear antigen in vitro. These findings provide new data on the biochemical activities of WRN that may help elucidate its role(s) in DNA metabolism. 相似文献
17.
Peter Burkovics Ildik Hajdú Valria Szukacsov Ildiko Unk Lajos Haracska 《Nucleic acids research》2009,37(13):4247-4255
Human Ape2 protein has 3′ phosphodiesterase activity for processing 3′-damaged DNA termini, 3′–5′ exonuclease activity that supports removal of mismatched nucleotides from the 3′-end of DNA, and a somewhat weak AP-endonuclease activity. However, very little is known about the role of Ape2 in DNA repair processes. Here, we examine the effect of interaction of Ape2 with proliferating cell nuclear antigen (PCNA) on its enzymatic activities and on targeting Ape2 to oxidative DNA lesions. We show that PCNA strongly stimulates the 3′–5′ exonuclease and 3′ phosphodiesterase activities of Ape2, but has no effect on its AP-endonuclease activity. Moreover, we find that upon hydrogen-peroxide treatment Ape2 redistributes to nuclear foci where it colocalizes with PCNA. In concert with these results, we provide biochemical evidence that Ape2 can reduce the mutagenic consequences of attack by reactive oxygen species not only by repairing 3′-damaged termini but also by removing 3′-end adenine opposite from 8-oxoG. Based on these findings we suggest the involvement of Ape2 in repair of oxidative DNA damage and PCNA-dependent repair synthesis. 相似文献
18.
Most eukaryotic DNA replication is performed by A- and B-family DNA polymerases which possess a faithful polymerase activity that preferentially incorporates correct over incorrect nucleotides. Additionally, many replicative polymerases have an efficient 3′→5′ exonuclease activity that excises misincorporated nucleotides. Together, these activities contribute to overall low polymerase error frequency (one error per 106–108 incorporations) and support faithful eukaryotic genome replication. Eukaryotic DNA polymerase ϵ (Polϵ) is one of three main replicative DNA polymerases for nuclear genomic replication and is responsible for leading strand synthesis. Here, we employed pre-steady-state kinetic methods and determined the overall fidelity of human Polϵ (hPolϵ) by measuring the individual contributions of its polymerase and 3′→5′ exonuclease activities. The polymerase activity of hPolϵ has a high base substitution fidelity (10−4–10−7) resulting from large decreases in both nucleotide incorporation rate constants and ground-state binding affinities for incorrect relative to correct nucleotides. The 3′→5′ exonuclease activity of hPolϵ further enhances polymerization fidelity by an unprecedented 3.5 × 102 to 1.2 × 104-fold. The resulting overall fidelity of hPolϵ (10−6–10−11) justifies hPolϵ to be a primary enzyme to replicate human nuclear genome (0.1–1.0 error per round). Consistently, somatic mutations in hPolϵ, which decrease its exonuclease activity, are connected with mutator phenotypes and cancer formation. 相似文献
19.
XPA plays an important role in the DNA damage recognition during human nucleotide excision repair. Here we report that the XPA is a homodimer either in the free state or as a complex with human RPA in solution under normal conditions. The human XPA protein purified from baculovirus-infected sf21 insect cells has a molecular mass of 36 317 Da, as determined by mass spectroscopy. However, the apparent molecular mass of XPA determined by the native gel filtration chromatography was about 71 kDa, suggesting that XPA is a dimer. This observation was supported by a native PFO-PAGE and fluorescence spectroscopy analysis. XPA formed a dimer (XPA2) in a broad range of XPA and NaCl concentrations, and the dimerization was not due to the disulfide bond formation. Furthermore, a titration analysis of the binding of XPA to the human RPA indicated that it was the XPA2 that formed the complex with RPA. Finally, the difference between the mass spectrometric and the calculated masses of XPA implies that the protein contains posttranslational modifications. Taken together, our data suggest that the dimerization of XPA may play an important role in the DNA damage recognition of nucleotide excision repair. 相似文献
20.
Phi29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein
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Phage Φ29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as the primer for initiation of DNA synthesis. In one of the most important motifs present in the 3′→5′ exonucleolytic domain of proofreading DNA polymerases, the ExoII motif, Φ29 DNA polymerase contains three amino acid residues, Y59, H61 and F69, which are highly conserved among most proofreading DNA polymerases. These residues have recently been shown to be involved in proper stabilization of the primer terminus at the 3′→5′ exonuclease active site. Here we investigate by means of site-directed mutagenesis the role of these three residues in reactions that are specific for DNA polymerases utilizing a protein-primed DNA replication mechanism. Mutations introduced at residues Y59, H61 and F69 severely affected the protein-primed replication capacity of Φ29 DNA polymerase. For four of the mutants, namely Y59L, H61L, H61R and F69S, interaction with the terminal protein was affected, leading to few initiation and transition products. These findings, together with the specific conservation of Y59, H61 and F69 among DNA polymerases belonging to the protein-primed subgroup, strongly suggest a functional role of these amino acid residues in the DNA polymerase–terminal protein interaction. 相似文献