Abasic (AP) sites are one of the most frequently formed lesions in DNA, and they present a strong block to continued synthesis by the replicative DNA machinery. Here we show efficient bypass of an AP site by the combined action of yeast DNA polymerases ␦ and . In this reaction, Pol␦ inserts an A nucleotide opposite the AP site, and Pol subsequently extends from the inserted nucleotide. Consistent with these observations, sequence analyses of mutations in the yeast CAN1 s gene indicate that A is the nucleotide inserted most often opposite AP sites. The nucleotides C, G, and T are also incorporated, but much less frequently. Enzymes such as Rev1 and Pol may contribute to the insertion of these other nucleotides; the predominant role of Rev1 in AP bypass, however, is likely to be structural. Steady-state kinetic analyses show that Pol is highly inefficient in incorporating nucleotides opposite the AP site, but it efficiently extends from nucleotides, particularly an A, inserted opposite this lesion. Thus, in eukaryotes, bypass of an AP site requires the sequential action of two DNA polymerases, wherein the extension step depends solely upon Pol, but the insertion step can be quite varied, involving not only the predominant action of the replicative DNA polymerase, Pol␦, but also the less prominent role of various translesion synthesis polymerases. Abasic (AP) sites represent one of the most frequently formed DNA lesions in eukaryotes, and it has been estimated that a human cell loses as many as 10 4 purines per day from its genome (Lindahl and Nyberg 1972). In Saccharomyces cerevisiae, AP sites are efficiently repaired by the AP endonucleases encoded by the APN1 and APN2 genes. APN1 and APN2 provide alternate pathways for the removal of AP sites, and consequently, simultaneous inactivation of both the genes results in a dramatic decline in the efficiency to repair AP sites (Johnson et al. 1998).If the AP sites are not removed by excision repair processes, they present a block to the replication machinery. During replication, AP sites can be bypassed either by a specialized mutagenic DNA polymerase, or by errorfree mechanisms such as recombination or a copy-choice type of DNA synthesis. In S. cerevisiae, genes in the RAD6 epistasis group promote replication through DNA lesions (Prakash 1981). The REV1, REV3, and REV7 genes of this group are essential for UV-induced mutagenesis (Lawrence and Hinkle 1996), and these genes are also indispensable for mutagenesis induced by AP sites (Johnson et al. 1998). REV1 encodes a deoxycytidyl transferase activity (Nelson et al. 1996a), and the REV3-and REV7-encoded proteins together form DNA polymerase (Nelson et al. 1996b). Although Pol is absolutely required for damage-induced mutagenesis, and therefore for the mutagenic bypass of a variety of DNA lesions, our recent studies have indicated that on its own, Pol bypasses UV lesions very inefficiently (Johnson et al. 2000a). This is because Pol is very inefficient in inserting nucleotides opposite the 3Ј T of a cis-syn thymine-thym...

SummaryLesions in the template DNA strand block the progression of the replication fork. In the yeast Saccharomyces cerevisiae, replication through DNA lesions is mediated by different Rad6-Rad18-dependent means, which include translesion synthesis and a Rad5-dependent postreplicational repair pathway that repairs the discontinuities that form in the DNA synthesized from damaged templates. Although translesion synthesis is well characterized, little is known about the mechanisms that modulate Rad5-dependent postreplicational repair. Here we show that yeast Rad5 has a DNA helicase activity that is specialized for replication fork regression. On model replication fork structures, Rad5 concertedly unwinds and anneals the nascent and the parental strands without exposing extended single-stranded regions. These observations provide insight into the mechanism of postreplicational repair in which Rad5 action promotes template switching for error-free damage bypass.

Human helicase-like transcription factor (HLTF) is frequently inactivated in colorectal and gastric cancers. Here, we show that HLTF is a functional homologue of yeast Rad5 that promotes error-free replication through DNA lesions. HLTF and Rad5 share the same unique structural features, including a RING domain embedded within a SWI/SNF helicase domain and an HIRAN domain. We find that inactivation of HLTF renders human cells sensitive to UV and other DNA-damaging agents and that HLTF complements the UV sensitivity of a rad5⌬ yeast strain. Also, similar to Rad5, HLTF physically interacts with the Rad6 -Rad18 and Mms2-Ubc13 ubiquitin-conjugating enzyme complexes and promotes the Lys-63-linked polyubiquitination of proliferating cell nuclear antigen at its Lys-164 residue. A requirement of HLTF for error-free postreplication repair of damaged DNA is in keeping with its cancersuppression role.yeast Rad5 ͉ damage bypass ͉ K63 polyubiquitination ͉ tumor suppressor L esions in DNA impose a block to synthesis by the replicative polymerases (Pols), and unless replication is rescued by the timely action of lesion bypass processes, stalled replication forks can collapse, leading to genomic instability. In eukaryotes the Rad6-Rad18 enzyme complex regulates lesion bypass processes that ensure the completion of replication. Rad6, a ubiquitinconjugating enzyme, forms a tight complex with Rad18, a RING-finger type ubiquitin ligase that binds DNA (1, 2), and in cells treated with DNA-damaging agents, Rad6-Rad18 monoubiquitinates proliferating cell nuclear antigen (PCNA), a DNA Pol sliding clamp that is a key component of the replication machinery (3). Ubiquitination at the Lys-164 residue of PCNA and its subsequent polyubiquitination serves as a molecular switch between various DNA damage bypass processes (3-5).In the yeast Saccharomyces cerevisiae, Rad6-Rad18 governs at least three alternative pathways for promoting replication through DNA lesions (6). Two pathways activated by PCNA monoubiquitination are carried out by specialized translesion synthesis (TLS) DNA Pols, such as Pol and Pol , which are able to copy DNA directly from the damaged template on an error-free or error-prone way (6). The third pathway called postreplication repair (PRR), however, is activated by PCNA polyubiquitination and operates by template switching using the information of the undamaged newly synthesized nascent strand on the sister duplex for DNA synthesis across damaged DNA (7-10). The PRR pathway depends on the Rad5, Mms2, and Ubc13 proteins and promotes error-free replication through DNA lesions. Recently, we have shown that yeast Rad5 has a DNA helicase activity that is specialized for replication fork regression, as Rad5 can concertedly unwind and anneal the nascent and the parental strands of the fork without exposing any single-stranded regions (7). This Rad5 activity would ensure damage bypass by promoting replication fork regression where the newly synthesized DNA strand of the sister duplex can be used as a template. In addition to its r...

In both yeast and humans, DNA polymerase (Pol) eta functions in error-free replication of ultraviolet-damaged DNA, and Poleta promotes replication through many other DNA lesions as well. Here, we present evidence for the physical and functional interaction of yeast Poleta with proliferating cell nuclear antigen (PCNA) and show that the interaction with PCNA is essential for the in vivo function of Poleta. Poleta is highly inefficient at inserting a nucleotide opposite an abasic site, but interaction with PCNA greatly stimulates its ability for nucleotide incorporation opposite this lesion. Thus, in addition to having a pivotal role in the targeting of Poleta to the replication machinery stalled at DNA lesions, interaction with PCNA would promote the bypass of certain DNA lesions.

Human DNA polymerase (hPol) functions in the error-free replication of UV-damaged DNA, and mutations in hPol cause cancer-prone syndrome, the variant form of xeroderma pigmentosum. However, in spite of its key role in promoting replication through a variety of distorting DNA lesions, the manner by which hPol is targeted to the replication machinery stalled at a lesion site remains unknown. Here, we provide evidence for the physical interaction of hPol with proliferating cell nuclear antigen (PCNA) and show that mutations in the PCNA binding motif of hPol inactivate this interaction. PCNA, together with replication factor C and replication protein A, stimulates the DNA synthetic activity of hPol, and steady-state kinetic studies indicate that this stimulation accrues from an increase in the efficiency of nucleotide insertion resulting from a reduction in the apparent K m for the incoming nucleotide.DNA polymerase (Pol) is unique among eukaryotic DNA polymerases in its proficient ability to replicate through distorting DNA lesions. Both in yeast and in humans, Pol functions in the error-free replication of UV-damaged DNA (19,26,34,39), and mutations in human Pol (hPol) result in cancer-prone syndrome, the variant form of xeroderma pigmentosum (XP-V) (17, 25). Interestingly, both yeast Pol and hPol replicate through a cis-syn thymine-thymine (TT) dimer with the same efficiency and accuracy as they replicate through undamaged T's (18, 21, 37). Also, genetic studies with yeast have indicated a role for Pol in the error-free bypass of cyclobutane pyrimidine dimers that are formed at 5Ј-TC-3Ј and 5Ј-CC-3Ј sites (40). Pol also promotes replication through a (6-4) TT photoproduct, a highly distorting DNA lesion, by preferentially inserting a G residue opposite the 3Ј T of the photoproduct. Subsequently, Pol efficiently promotes extension from the G residue by inserting the correct nucleotide, A, opposite the 5Ј T of the lesion (16). Although the insertion of a G opposite the 3Ј T of the (6-4) TT photoproduct would cause 3Ј T3C substitutions, it was previously suggested that a similar insertion of G by Pol opposite the 3Ј C of the 5Ј-TC-3Ј and 5Ј-CC-3Ј (6-4) photoproducts, followed by extension by Pol by the insertion of the correct nucleotide opposite the 5Ј residue of this lesion, would lead to error-free bypass of the DNA lesion (16). Since (6-4) photoproducts are formed much more frequently at TC and CC sites than at TT sites (4, 6), Pol would largely contribute to the error-free bypass of (6-4) lesions as well. Yeast Pol and hPol also efficiently replicate through other DNA lesions, such as 8-oxoguanine (15) and O 6 -methylguanine (13).The ability of Pol to replicate through distorting DNA lesions has suggested that the active site of Pol is tolerant of geometric distortions introduced into DNA by these lesions. As a consequence, Pol is a low-fidelity enzyme, and on undamaged DNA, the yeast and human enzymes misincorporate nucleotides with a frequency of 10 Ϫ2 to 10 Ϫ3 (21, 38). In sharp contrast, replicative DNA pol...

No abstract

Human SHPRH gene is located at the 6q24 chromosomal region, and loss of heterozygosity in this region is seen in a wide variety of cancers. SHPRH is a member of the SWI͞SNF family of ATPases͞ helicases, and it possesses a C 3HC4 RING motif characteristic of ubiquitin ligase proteins. In both of these features, SHPRH resembles the yeast Rad5 protein, which, together with Mms2-Ubc13, promotes replication through DNA lesions via an error-free postreplicational repair pathway. Genetic evidence in yeast has indicated a role for Rad5 as a ubiquitin ligase in mediating the Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Here we show that SHPRH is a functional homolog of Rad5. Similar to Rad5, SHPRH physically interacts with the Rad6 -Rad18 and Mms2-Ubc13 complexes, and we show that SHPRH protein is a ubiquitin ligase indispensable for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Based on these observations, we predict a role for SHPRH in promoting error-free replication through DNA lesions. Such a role for SHPRH is consistent with the observation that this gene is mutated in a number of cancer cell lines, including those from melanomas and ovarian cancers, which raises the strong possibility that SHPRH function is an important deterrent to mutagenesis and carcinogenesis in humans.postreplication repair ͉ translesion synthesis ͉ tumor suppressor

Unrepaired DNA lesions can block the progression of the replication fork, leading to genomic instability and cancer in higher-order eukaryotes. In Saccharomyces cerevisiae, replication through DNA lesions can be mediated by translesion synthesis DNA polymerases, leading to error-free or error-prone damage bypass, or by Rad5-mediated template switching to the sister chromatid that is inherently error free. While translesion synthesis pathways are highly conserved from yeast to humans, very little is known of a Rad5-like pathway in human cells. Here we show that a human homologue of Rad5, HLTF, can facilitate fork regression and has a role in replication of damaged DNA. We found that HLTF is able to reverse model replication forks, a process which depends on its double-stranded DNA translocase activity. Furthermore, from analysis of isolated dually labeled chromosomal fibers, we demonstrate that in vivo, HLTF promotes the restart of replication forks blocked at DNA lesions. These findings suggest that HLTF can promote error-free replication of damaged DNA and support a role for HLTF in preventing mutagenesis and carcinogenesis, providing thereby for its potential tumor suppressor role.Genomic instability underlies the development of various human diseases, including cancer. Cancer genomes are highly heterogeneous and can possess various instability phenotypes, including accumulation of gross chromosomal rearrangements (GCR) (9, 40). A plethora of evidence has indicated that defects in various DNA repair pathways promote genomic instability and trigger subsequent tumor formation (1). One example is the mutational or epigenetic inactivation of the mismatch repair gene MSH2 or MLH1 in a subset of colorectal cancers (4, 39). An apparently distinct subset of colon cancers, representing about 40% of malignant colorectal transformations, is characterized by epigenetic inactivation of the HLTF gene (22). While HLTF has been suggested to act as a transcription factor (31), recent studies indicated a role for HLTF in replication of damaged DNA, raising the possibility that it is this function of HLTF that can lead to suppression of genomic instability (23, 37).In Saccharomyces cerevisiae, genetic data have indicated a crucial role for the Rad6-Rad18 protein complex, Rad5, and the Mms2-Ubc13 complex in the replication of damaged DNA (26). In contrast to nucleotide incorporation opposite the lesion by specialized translesion synthesis polymerases, which requires Rad6-Rad18-dependent monoubiquitylation of PCNA, template-switching-mediated bypass depends on Lys63 polyubiquitylation of PCNA by the Mms2-Ubc13 ubiquitin-conjugating enzyme complex and Rad5 ubiquitin ligase (10,12,36,41). Yeast genetic data have shown that not only the ubiquitin ligase activity of Rad5 but also its ATPase activity is essential for its function in replication of damaged DNA (5). In agreement with the in vivo data, Rad5 is an ATP-hydrolysis-driven molecular motor which can facilitate template switching at stalled replication forks (2). While translesi...