DNA Polymerase ε: A Polymerase of Unusual Size (and Complexity)

Single-stranded DNA-binding protein in Bacteria and replication protein A (RPA) in Eukarya play crucial roles in DNA replication, repair, and recombination processes. We identified an RPA complex from the hyperthermophilic archaeon, Pyrococcus furiosus. Unlike the single-peptide RPAs from the methanogenic archaea, Methanococcus jannaschii and Methanothermobacter thermoautotrophicus, P. furiosus RPA (PfuRPA) exists as a stable hetero-oligomeric complex consisting of three subunits, RPA41, RPA14, and RPA32. The amino acid sequence of RPA41 has some similarity to those of the eukaryotic RPA70 subunit and the M. jannaschii RPA. On the other hand, RPA14 and RPA32 do not share homology with any known open reading frames from Bacteria and Eukarya. However, six of eight archaea, whose total genome sequences have been published, have the open reading frame homologous to RPA32. The PfuRPA complex, but not each subunit alone, specifically bound to a single-stranded DNA and clearly enhanced the efficiency of an in vitro strand-exchange reaction by the P. furiosus RadA protein. Moreover, immunoprecipitation analyses showed that PfuRPA interacts with the recombination proteins, RadA and Hjc, as well as replication proteins, DNA polymerases, primase, proliferating cell nuclear antigen, and replication factor C in P. furiosus cells. These results indicate that PfuRPA plays important roles in the homologous DNA recombination in P. furiosus.

DNA ligases catalyse the joining of single and double-strand DNA breaks, which is an essential final step in DNA replication, recombination and repair. Mammalian cells have four DNA ligases, termed ligases I-IV. In contrast, other than a DNA ligase I homologue (encoded by CDC9), no other DNA ligases have hitherto been identified in Saccharomyces cerevisiae. Here, we report the identification and characterization of a novel gene, LIG4, which encodes a protein with strong homology to mammalian DNA ligase IV. Unlike CDC9, LIG4 is not essential for DNA replication, RAD52-dependent homologous recombination nor the repair of UV light-induced DNA damage. Instead, it encodes a crucial component of the non-homologous end-joining (NHEJ) apparatus, which repairs DNA double-strand breaks that are generated by ionizing radiation or restriction enzyme digestion: a function which cannot be complemented by CDC9. Lig4p acts in the same DNA repair pathway as the DNA end-binding protein Ku. However, unlike Ku, it does not function in telomere length homeostasis. These findings indicate diversification of function between different eukaryotic DNA ligases. Furthermore, they provide insights into mechanisms of DNA repair and suggest that the NHEJ pathway is highly conserved throughout the eukaryotic kingdom.

Widespread eukaryotic sequences, highly similar to bacterial DNA polymerase I, looking for functions

Two important and timely questions with respect to DNA replication, DNA recombination, and DNA repair are: (i) what controls which DNA polymerase gains access to a particular primer-terminus, and (ii) what determines whether a DNA polymerase hands off its DNA substrate to either a different DNA polymerase or to a different protein(s) for the completion of the specific biological process? These questions have taken on added importance in light of the fact that the number of known template-dependent DNA polymerases in both eukaryotes and in prokaryotes has grown tremendously in the past two years. Most notably, the current list now includes a completely new family of enzymes that are capable of replicating imperfect DNA templates. This UmuC-DinB-Rad30-Rev1 superfamily of DNA polymerases has members in all three kingdoms of life. Members of this family have recently received a great deal of attention due to the roles they play in translesion DNA synthesis (TLS), the potentially mutagenic replication over DNA lesions that act as potent blocks to continued replication catalyzed by replicative DNA polymerases. Here, we have attempted to summarize our current understanding of the regulation of action of DNA polymerases with respect to their roles in DNA replication, TLS, DNA repair, DNA recombination, and cell cycle progression. In particular, we discuss these issues in the context of the Gram-negative bacterium, Escherichia coli, that contains a DNA polymerase (Pol V) known to participate in most, if not all, of these processes.

DNA end resection has a key role in double-strand break repair and DNA replication. Defective DNA end resection can cause malfunctions in DNA repair and replication, leading to greater genomic instability. DNA end resection is initiated by MRN-CtIP generating short, 3′-single-stranded DNA (ssDNA). This newly generated ssDNA is further elongated by multiple nucleases and DNA helicases, such as EXO1, DNA2, and BLM. Effective DNA end resection is essential for error-free homologous recombination DNA repair, the degradation of incorrectly replicated DNA and double-strand break repair choice. Because of its importance in DNA repair, DNA end resection is strictly regulated. Numerous mechanisms have been reported to regulate the initiation, extension, and termination of DNA end resection. Here, we review the general process of DNA end resection and its role in DNA replication and repair pathway choice.

Many proteins involved in DNA replication and repair undergo post-translational modifications such as phosphorylation and ubiquitylation. Proliferating cell nuclear antigen (PCNA; a homotrimeric protein that encircles double-stranded DNA to function as a sliding clamp for DNA polymerases) is monoubiquitylated by the RAD6-RAD18 complex and further polyubiquitylated by the RAD5-MMS2-UBC13 complex in response to various DNA-damaging agents. PCNA mono- and polyubiquitylation activate an error-prone translesion synthesis pathway and an error-free pathway of damage avoidance, respectively. Here we show that replication factor C (RFC; a heteropentameric protein complex that loads PCNA onto DNA) was also ubiquitylated in a RAD18-dependent manner in cells treated with alkylating agents or H(2)O(2). A mutant form of RFC2 with a D228A substitution (corresponding to a yeast Rfc4 mutation that reduces an interaction with replication protein A (RPA), a single-stranded DNA-binding protein) was heavily ubiquitylated in cells even in the absence of DNA damage. Furthermore RFC2 was ubiquitylated by the RAD6-RAD18 complex in vitro, and its modification was inhibited in the presence of RPA. The inhibitory effect of RPA on RFC2 ubiquitylation was relatively specific because RAD6-RAD18-mediated ubiquitylation of PCNA was RPA-insensitive. Our findings suggest that RPA plays a regulatory role in DNA damage responses via repression of RFC2 ubiquitylation in human cells.

We have purified wild type and exonuclease-deficient four-subunit DNA polymerase epsilon (Pol epsilon) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes. Wild type Pol epsilon synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of </=2 x 10-5 and </=5 x 10-7, respectively. Pol epsilon lacking 3' --> 5' exonuclease activity is less accurate to a degree suggesting that wild type Pol epsilon proofreads at least 92% of base substitution errors and at least 99% of frameshift errors made by the polymerase. Surprisingly the base substitution fidelity of exonuclease-deficient Pol epsilon is severalfold lower than that of proofreading-deficient forms of other replicative polymerases. Moreover the spectrum of errors shows a feature not seen with other A, B, C, or X family polymerases: a high proportion of transversions resulting from T.dTTP, T.dCTP, and C.dTTP mispairs. This unique error specificity and amino acid sequence alignments suggest that the structure of the polymerase active site of Pol epsilon differs from those of other B family members. We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol epsilon in vitro and substitutions occurring in vivo in a yeast strain defective in Pol epsilon proofreading and DNA mismatch repair. We discuss the implications of these findings for the role of Pol epsilon polymerase activity in DNA replication.

The telomeric complex, shelterin, plays a critical role in protecting chromosome ends from erosion, and disruption of these complexes can lead to chromosomal instability culminating in cell death or malignant transformation. We reported previously that dominant-negative mutants of one of the telomeric proteins called TIN2 cause death of androgen receptor (AR)-negative but not AR-positive prostate cancer cells, raising the question of a possible role of AR in the structural stability of telomeric complexes. Consistent with this possibility, in the present study, we observed that the AR antagonist Casodex (bicalutamide) disrupted telomeric complexes in AR-positive LNCaP cells but not in AR-negative PC-3 cells. Immunofluorescent studies revealed colocalization of TIN2 and AR. Reciprocal immunoprecipitation studies showed association of AR with telomeric proteins. Furthermore, telomeric proteins were overexpressed in prostate cancer cells compared with normal prostate epithelial cells, and sucrose density gradient analysis showed co-sedimentation of AR with telomeric proteins in a shelterin-like mega complex. Together, these observations suggest an allosteric role of AR in telomere complex stability in prostate cancer cells and suggest that AR-antagonist Casodex-mediated cell death may be due to telomere complex disruption.

DNA polymerase epsilon (Pol epsilon) is a multi-subunit enzyme required for the initiation of chromosomal DNA replication. Here, we report the cloning of two fission yeast genes, called dpb3+ and dpb4+ that encode proteins homologous to the two smallest subunits of Pol epsilon. Although Dpb4 is not required for cell viability, Deltadpb4 mutants are synthetically lethal with mutations in four genes required for DNA replication initiation, cdc20+ (encoding DNA Pol epsilon), cut5+ (homologous to DPB11/TopBP1), sna41+ (homologous to CDC45) and cdc21+ (encoding Mcm4, a component of the pre-replicative complex). In contrast to Dpb4, Dpb3 is essential for cell cycle progression. A glutathione S-transferase pull-down assay indicates that Dpb3 physically interacts with both Dpb2 and Dpb4, suggesting that Dpb3 associates with other members of the Pol epsilon complex. Depletion of Dpb3 leads to an accumulation of cells in S phase consistent with Dpb3 having a role in DNA replication. In addition, many of the cells have a bi-nucleate or multinucleate phenotype, indicating that cell separation is also inhibited. Finally, we have examined in vivo localization of green fluorescent protein (GFP)-tagged Dpb3 and Dpb4 and found that both proteins are localized to the nucleus consistent with their proposed role in DNA replication. However, in the absence of Dpb3, GFP-Dpb4 appears to be more dispersed throughout the cell, suggesting that Dpb3 may be important in establishing or maintaining normal localization of Dpb4.

DNA polymerase epsilon (pol epsilon) has been implicated in DNA replication, DNA repair, and cell cycle control, but its precise roles are unclear. When the subcellular localization of human pol epsilon was examined by indirect immunofluorescence, pol epsilon appeared in discrete nuclear foci that colocalized with proliferating cell nuclear antigen (PCNA) foci and sites of DNA synthesis only late in S phase. Early in S phase, pol epsilon foci were adjacent to PCNA foci. In contrast to PCNA foci that were only present in S phase, pol epsilon foci were present throughout mitosis and the G(1) phase of cycling cells. It is hypothesized from these observations that pol epsilon and PCNA have separate but associated functions early in S phase and that pol epsilon participates with PCNA in DNA replication late in S phase.

DNA polymerase lambda (pol lambda) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol lambda null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol lambda in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol lambda co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol lambda protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.

Translesion DNA synthesis is an important branch of the DNA damage tolerance pathway that assures genomic integrity of living organisms. The mechanisms of DNA polymerase (Pol) switches during lesion bypass are not known. Here, we show that the C-terminal domain of the Pol ζ catalytic subunit interacts with accessory subunits of replicative DNA Pol δ. We also show that, unlike other members of the human B-family of DNA polymerases, the highly conserved and similar C-terminal domains of Pol δ and Pol ζ contain a [4Fe-4S] cluster coordinated by four cysteines. Amino acid changes in Pol ζ that prevent the assembly of the [4Fe-4S] cluster abrogate Pol ζ function in UV mutagenesis. On the basis of these data, we propose that Pol switches at replication-blocking lesions occur by the exchange of the Pol δ and Pol ζ catalytic subunits on a preassembled complex of accessory proteins retained on DNA during translesion DNA synthesis.

DNA polymerase epsilon (Pol epsilon) of Saccharomyces cerevisiae participates in many aspects of DNA replication, as well as in DNA repair. In order to clarify molecular mechanisms employed in the multiple tasks of Pol epsilon, we have been characterizing the interaction between Pol epsilon and DNA. Analysis of the four-subunit Pol epsilon complex by gel mobility shift assay revealed that the complex binds not only to single-stranded (ss) DNA but also equally well to double-stranded (ds) DNA. A truncated polypeptide consisting of the N-terminal domain of Pol2p catalytic subunit binds to ssDNA but not to dsDNA, indicating that the Pol2p C-terminal domain and/or the auxiliary subunits are involved in the dsDNA-binding. The dsDNA-binding by Pol epsilon does not require DNA ends or specific DNA sequences. Further analysis by competition experiments indicated that Pol epsilon contains at least two distinct DNA-binding sites, one of which binds exclusively to ssDNA and the other to dsDNA. The dsDNA-binding site, however, is suggested to also bind ssDNA. The DNA polymerase activity of Pol epsilon is inhibited by ssDNA but not by dsDNA. Furthermore, purification of the Pol epsilon auxiliary subunits Dpb3p and Dpb4p revealed that these proteins form a heterodimer and associate with dsDNA. Pol epsilon has multiple sites at which it interacts with DNA. One of these sites has a strong affinity for dsDNA, a feature that is not generally associated with DNA polymerases. Involvement of the Dpb3p-Dpb4p complex in the dsDNA-binding of Pol epsilon is inferred.

The isolation of DNA polymerase (Pol) epsilon from extracts of HeLa cells is described. The final fractions contained two major subunits of 210 and 50 kDa which cosedimented with Pol epsilon activity, similar to those described previously (Syvaoja, J., and Linn, S. (1989) J. Biol. Chem. 264, 2489-2497). The properties of the human Pol epsilon and the yeast Pol epsilon were compared. Both enzymes elongated singly primed single-stranded circular DNA templates. Yeast Pol epsilon required the presence of a DNA binding protein (SSB) whereas human Pol epsilon required the addition of SSB, Activator 1 and proliferating cell nuclear antigen (PCNA) for maximal activity. Both enzymes were totally unable to elongate primed DNA templates in the presence of salt; however, activity could be restored by the addition of Activator 1 and PCNA. Like Pol delta, Pol epsilon formed complexes with SSB-coated primed DNA templates in the presence of Activator 1 and PCNA which could be isolated by filtration through Bio-Gel A-5m columns. Unlike Pol delta, Pol epsilon bound to SSB-coated primed DNA in the absence of the auxiliary factors. In the presence of salt, Pol epsilon complexes were less stable than they were in the absence of salt. In the in vitro simian virus 40 (SV40) T antigen-dependent synthesis of DNA containing the SV40 origin of replication, yeast Pol epsilon but not human Pol epsilon could substitute for yeast or human Pol delta in the generation of long DNA products. However, human Pol epsilon did increase slightly the length of DNA chains formed by the DNA polymerase alpha-primase complex in SV40 DNA synthesis. The bearing of this observation on the requirement for a PCNA-dependent DNA polymerase in the synthesis and maturation of Okazaki fragments is discussed. However, no unique role for human Pol epsilon in the in vitro SV40 DNA replication system was detected.

Saccharomyces cerevisiae DNA polymerase δ (Pol δ) and DNA polymerase ε (Pol ε) are replicative DNA polymerases at the replication fork. Both enzymes are stimulated by PCNA, although to different levels. To understand why and to explore the interaction with PCNA, we compared Pol δ and Pol ε in physical interactions with PCNA and nucleic acids (with or without RPA), and in functional assays measuring activity and processivity. Using surface plasmon resonance technique, we show that Pol ε has a high affinity for DNA, but a low affinity for PCNA. In contrast, Pol δ has a low affinity for DNA and a high affinity for PCNA. The true processivity of Pol δ and Pol ε was measured for the first time in the presence of RPA, PCNA and RFC on single-stranded DNA. Remarkably, in the presence of PCNA, the processivity of Pol δ and Pol ε on RPA-coated DNA is comparable. Finally, more PCNA molecules were found on the template after it was replicated by Pol ε when compared to Pol δ. We conclude that Pol ε and Pol δ exhibit comparable processivity, but are loaded on the primer-end via different mechanisms.