跨损伤合成的DNA聚合酶——一类新的DNA聚合酶

细胞虽然拥有多种修复途径,但有些DNA损伤仍不可避免地会逃避修复而在基因组上保留下来,细胞跨损伤DNA合成的分子机制一直是DNA修复中主要的未解决问题之一.最近通过对一类结构相关性UmuC/DinB蛋白质超家族成员的研究发现它们具有DNA聚合酶功能.这类新发现的DNA聚合酶不同于经典的复制性DNA聚合酶,它们能以易误/突变(error-prone/mutagenic)或无误(error-free)方式进行跨损伤(translesion)DNA合成,并且从细菌到人在进化上功能保守.     

DNA synthesis across an abasic lesion by yeast REV1 DNA polymerase

Abasic (apurinic/apyrimidinic) sites are among the most abundant DNA lesions in humans, and they present a strong block to replication. They are also highly mutagenic because when replicative DNA polymerases manage to insert a nucleotide opposite the lesion, they prefer to insert an A. Rev1, a member of Y-family DNA polymerases, does not obey the A-rule. This enzyme inserts a C opposite an abasic lesion with much greater catalytic efficiency than an A, G, or T. We present here the structure of yeast Rev1 in ternary complex with DNA containing an abasic lesion and with dCTP as the incoming nucleotide. The structure reveals a mechanism of synthesis across an abasic lesion that differs from that in other polymerases. The lesion is driven to an extrahelical position, and the incorporation of a C is mediated by an arginine (Arg324) that is conserved in all known orthologs of Rev1, including humans. The hydrophobic cavity that normally accommodates the unmodified G is instead filled with water molecules. Since Gs are especially prone to depurination through a spontaneous hydrolysis of the glycosidic bond, the ability of Rev1 to stabilize an abasic lesion in its active site and employ a surrogate arginine to incorporate a C provides a unique means for the “error-free” bypass of this noninstructional lesion.     

Translesion synthesis across abasic lesions by human B-family and Y-family DNA polymerases α, δ, η, ι, κ, and REV1

Abasic (apurinic/apyrimidinic, AP) sites are the most common DNA lesions formed in cells, induce severe blocks to DNA replication, and are highly mutagenic. Human Y-family translesion DNA polymerases (pols) such as pols η, ι, κ, and REV1 have been suggested to play roles in replicative bypass across many DNA lesions where B-family replicative pols stall, but their individual catalytic functions in AP site bypass are not well understood. In this study, oligonucleotides containing a synthetic abasic lesion (tetrahydrofuran analogue) were compared for catalytic efficiency and base selectivity with human Y-family pols η, ι, κ, and REV1 and B-family pols α and δ. Pol η and pol δ/proliferating cell nuclear antigen (PCNA) copied past AP sites quite effectively and generated products ranging from one-base to full-length extension. Pol ι and REV1 readily incorporated one base opposite AP sites but then stopped. Pols κ and α were severely blocked at AP sites. Pol η preferentially inserted T and A; pol ι inserted T, G, and A; pol κ inserted C and A; REV1 preferentially inserted C opposite AP sites. The B-family pols α and δ/PCNA preferentially inserted A (85% and 58%, respectively) consonant with the A-rule hypothesis. Pols η and δ/PCNA were much more efficient in next-base extension, preferably from A positioned opposite an AP site, than pol κ. These results suggest that AP sites might be bypassed with moderate efficiency by single B- and Y-family pols or combinations, possibly by REV1 and pols ι, η, and δ/PCNA at the insertion step opposite the lesion and by pols η and δ/PCNA at the subsequent extension step. The patterns of the base preferences of human B-family and Y-family pols in both insertion and extension are pertinent to some of the mutagenesis events induced by AP lesions in human cells.     

Lesion (in)tolerance reveals insights into DNA replication fidelity

The initial encounter of an unrepaired DNA lesion is likely to be with a replicative DNA polymerase, and the outcome of this event determines whether an error-prone or error-free damage avoidance pathway is taken. To understand the atomic details of this critical encounter, we have determined the crystal structures of the pol alpha family RB69 DNA polymerase with DNA containing the two most prevalent, spontaneously generated premutagenic lesions, an abasic site and 2'-deoxy-7,8-dihydro-8-oxoguanosine (8-oxodG). Identification of the interactions between these damaged nucleotides and the active site provides insight into the capacity of the polymerase to incorporate a base opposite the lesion. A novel open, catalytically inactive conformation of the DNA polymerase has been identified in the complex with a primed abasic site template. This structure provides the first molecular characterization of the DNA synthesis barrier caused by an abasic site and suggests a general mechanism for polymerase fidelity. In contrast, the structure of the ternary 8-oxodG:dCTP complex is almost identical to the replicating complex containing unmodified DNA, explaining the relative ease and fidelity by which this lesion is bypassed.     

Shared Genetic Pathways Contribute to the Tolerance of Endogenous and Low-Dose Exogenous DNA Damage in Yeast

DNA damage that escapes repair and blocks replicative DNA polymerases is tolerated by bypass mechanisms that fall into two general categories: error-free template switching and error-prone translesion synthesis. Prior studies of DNA damage responses in Saccharomyces cerevisiae have demonstrated that repair mechanisms are critical for survival when a single, high dose of DNA damage is delivered, while bypass/tolerance mechanisms are more important for survival when the damage level is low and continuous (acute and chronic damage, respectively). In the current study, epistatic interactions between DNA-damage tolerance genes were examined and compared when haploid yeast cells were exposed to either chronic ultraviolet light or chronic methyl methanesulfonate. Results demonstrate that genes assigned to error-free and error-prone bypass pathways similarly promote survival in the presence of each type of chronic damage. In addition to using defined sources of chronic damage, rates of spontaneous mutations generated by the Pol ζ translesion synthesis DNA polymerase (complex insertions in a frameshift-reversion assay) were used to infer epistatic interactions between the same genes. Similar epistatic interactions were observed in analyses of spontaneous mutation rates, suggesting that chronic DNA-damage responses accurately reflect those used to tolerate spontaneous lesions. These results have important implications when considering what constitutes a safe and acceptable level of exogenous DNA damage.     

Genetic Control of Replication through N1-methyladenine in Human Cells

N1-methyl adenine (1-MeA) is formed in DNA by reaction with alkylating agents and naturally occurring methyl halides. The 1-MeA lesion impairs Watson-Crick base pairing and blocks normal DNA replication. Here we identify the translesion synthesis (TLS) DNA polymerases (Pols) required for replicating through 1-MeA in human cells and show that TLS through this lesion is mediated via three different pathways in which Pols ι and θ function in one pathway and Pols η and ζ, respectively, function in the other two pathways. Our biochemical studies indicate that in the Polι/Polθ pathway, Polι would carry out nucleotide insertion opposite 1-MeA from which Polθ would extend synthesis. In the Polη pathway, this Pol alone would function at both the nucleotide insertion and extension steps of TLS, and in the third pathway, Polζ would extend from the nucleotide inserted opposite 1-MeA by an as yet unidentified Pol. Whereas by pushing 1-MeA into the syn conformation and by forming Hoogsteen base pair with the T residue, Polι would carry out TLS opposite 1-MeA, the ability of Polη to replicate through 1-MeA suggests that despite its need for Watson-Crick hydrogen bonding, Polη can stabilize the adduct in its active site. Remarkably, even though Pols η and ι are quite error-prone at inserting nucleotides opposite 1-MeA, TLS opposite this lesion in human cells occurs in a highly error-free fashion. This suggests that the in vivo fidelity of TLS Pols is regulated by factors such as post-translational modifications, protein-protein interactions, and possibly others.     

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance （DDT） has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen （PCNA） by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis （TLS） with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer.     

Structural insight into dynamic bypass of the major cisplatin‐DNA adduct by Y‐family polymerase Dpo4

Y‐family DNA polymerases bypass Pt‐GG, the cisplatin‐DNA double‐base lesion, contributing to the cisplatin resistance in tumour cells. To reveal the mechanism, we determined three structures of the Y‐family DNA polymerase, Dpo4, in complex with Pt‐GG DNA. The crystallographic snapshots show three stages of lesion bypass: the nucleotide insertions opposite the 3′G (first insertion) and 5′G (second insertion) of Pt‐GG, and the primer extension beyond the lesion site. We observed a dynamic process, in which the lesion was converted from an open and angular conformation at the first insertion to a depressed and nearly parallel conformation at the subsequent reaction stages to fit into the active site of Dpo4. The DNA translocation‐coupled conformational change may account for additional inhibition on the second insertion reaction. The structures illustrate that Pt‐GG disturbs the replicating base pair in the active site, which reduces the catalytic efficiency and fidelity. The in vivo relevance of Dpo4‐mediated Pt‐GG bypass was addressed by a dpo‐4 knockout strain of Sulfolobus solfataricus, which exhibits enhanced sensitivity to cisplatin and proteomic alterations consistent with genomic stress.     

DNA polymerase beta can incorporate ribonucleotides during DNA synthesis of undamaged and CPD-damaged DNA

Overexpression of the error-prone DNA polymerase beta (Pol beta) has been found to increase spontaneous mutagenesis by competing with the replicative polymerases during DNA replication. Here, we investigate an additional mechanism potentially used by Pol beta to enhance genetic instability via its ability to incorporate ribonucleotides into DNA. By using an in vitro primer extension assay, we show that purified human and calf thymus Pol beta can synthesize up to 8-mer long RNA. Moreover, Pol beta can efficiently incorporate rCTP opposite G in the absence of dCTP and, to a lesser extent, rATP opposite T in the absence of dATP and rGTP opposite C in the absence of dGTP. Recently, Pol beta was shown to catalyze in vitro translesion replication of a thymine cyclobutane pyrimidine dimer (CPD). Here, we investigate if ribonucleotides could be incorporated opposite the CPD damage and modulate the efficiency of the bypass process. We find that all four rNTPs can be incorporated opposite the CPD lesion, and that this process affects translesion synthesis. We discuss how incorporation of ribonucleotides into DNA may contribute to the high frequency of mutagenesis observed in Pol beta up-regulating cells.     

The Role of Dbf4-Dependent Protein Kinase in DNA Polymerase ζ-Dependent Mutagenesis in Saccharomyces cerevisiae

The yeast Dbf4-dependent kinase (DDK) (composed of Dbf4 and Cdc7 subunits) is an essential, conserved Ser/Thr protein kinase that regulates multiple processes in the cell, including DNA replication, recombination and induced mutagenesis. Only DDK substrates important for replication and recombination have been identified. Consequently, the mechanism by which DDK regulates mutagenesis is unknown. The yeast mcm5-bob1 mutation that bypasses DDK’s essential role in DNA replication was used here to examine whether loss of DDK affects spontaneous as well as induced mutagenesis. Using the sensitive lys2ΔA746 frameshift reversion assay, we show DDK is required to generate “complex” spontaneous mutations, which are a hallmark of the Polζ translesion synthesis DNA polymerase. DDK co-immunoprecipitated with the Rev7 regulatory, but not with the Rev3 polymerase subunit of Polζ. Conversely, Rev7 bound mainly to the Cdc7 kinase subunit and not to Dbf4. The Rev7 subunit of Polζ may be regulated by DDK phosphorylation as immunoprecipitates of yeast Cdc7 and also recombinant Xenopus DDK phosphorylated GST-Rev7 in vitro. In addition to promoting Polζ-dependent mutagenesis, DDK was also important for generating Polζ-independent large deletions that revert the lys2ΔA746 allele. The decrease in large deletions observed in the absence of DDK likely results from an increase in the rate of replication fork restart after an encounter with spontaneous DNA damage. Finally, nonepistatic, additive/synergistic UV sensitivity was observed in cdc7Δ pol32Δ and cdc7Δ pol30-K127R,K164R double mutants, suggesting that DDK may regulate Rev7 protein during postreplication “gap filling” rather than during “polymerase switching” by ubiquitinated and sumoylated modified Pol30 (PCNA) and Pol32.     

Architecture of the Pol III–clamp–exonuclease complex reveals key roles of the exonuclease subunit in processive DNA synthesis and repair

DNA polymerase III (Pol III) is the catalytic α subunit of the bacterial DNA Polymerase III holoenzyme. To reach maximum activity, Pol III binds to the DNA sliding clamp β and the exonuclease ε that provide processivity and proofreading, respectively. Here, we characterize the architecture of the Pol III–clamp–exonuclease complex by chemical crosslinking combined with mass spectrometry and biochemical methods, providing the first structural view of the trimeric complex. Our analysis reveals that the exonuclease is sandwiched between the polymerase and clamp and enhances the binding between the two proteins by providing a second, indirect, interaction between the polymerase and clamp. In addition, we show that the exonuclease binds the clamp via the canonical binding pocket and thus prevents binding of the translesion DNA polymerase IV to the clamp, providing a novel insight into the mechanism by which the replication machinery can switch between replication, proofreading, and translesion synthesis.     

Multiple functions of DNA polymerases

The primary role of DNA polymerases is to accurately and efficiently replicate the genome in order to ensure the maintenance of the genetic information and its faithful transmission through generations. This is not a simple task considering the size of the genome and its constant exposure to endogenous and environmental DNA damaging agents. Thus, a number of DNA repair pathways operate in cells to protect the integrity of the genome. In addition to their role in replication, DNA polymerases play a central role in most of these pathways. Given the multitude and the complexity of DNA transactions that depend on DNA polymerase activity, it is not surprising that cells in all organisms contain multiple highly specialized DNA polymerases, the majority of which have only recently been discovered. Five DNA polymerases are now recognized in Escherichia coli, 8 in Saccharomyces cerevisiae, and at least 15 in humans. While polymerases in bacteria, yeast and mammalian cells have been extensively studied much less is known about their counterparts in plants. For example, the plant model organism Arabidopsis thaliana is thought to contain 12 DNA polymerases, whose functions are mostly unknown. Here we review the properties and functions of DNA polymerases focusing on yeast and mammalian cells but paying special attention to the plant enzymes and the special circumstances of replication and repair in plant cells.     

Interaction between DNA polymerase λ and RPA during translesion synthesis

Replication of damaged DNA (translesion synthesis, TLS) is realized by specialized DNA polymerases. Additional protein factors such as replication protein A (RPA) play important roles in this process. However, details of the interaction are unknown. Here we analyzed the influence of the hRPA and its mutant hABCD lacking domains responsible for protein-protein interactions on ability of DNA polymerase lambda to catalyze TLS. The primer-template structures containing varying parts of extended strand (16 and 37 nt) were used as model systems imitating DNA intermediate of first stage of TLS. The 8-oxoguanine disposed in +1 position of the template strand in relation to 3 -end of primer was exploited as damage. It was shown that RPA stimulated TLS DNA synthesis catalyzed by DNA polymerase lambda in its globular but not in extended conformation. Moreover, this effect is dependent on the presence of p70N and p32C domains in RPA molecule.     

REV7 is required for anaphase-promoting complex-dependent ubiquitination and degradation of translesion DNA polymerase REV1

REV1 is a Y-family polymerase specialized for replicating across DNA lesions at the stalled replication folk. Due to the high error rate of REV1-dependent translesion DNA synthesis (TLS), tight regulation of REV1 activity is essential. Here, we show that human REV1 undergoes proteosomal degradation mediated by the E3 ubiquitin ligase known as anaphase-promoting complex (APC). REV1 associates with APC. Overexpression of APC coactivator CDH1 or CDC20 promotes polyubiquitination and proteosomal degradation of REV1. Surprisingly, polyubiquitination of REV1 also requires REV7, a TLS accessory protein that interacts with REV1 and other TLS polymerases. The N-terminal region of REV1 contains both the APC degron and an additional REV7-binding domain. Depletion of REV7 by RNA interference stabilizes REV1 by preventing polyubiquitination, whereas overexpression of REV7 augments REV1 degradation. Taken together, our findings suggest a role of REV7 in governing REV1 stability and interplay between TLS and APC-dependent proteolysis.