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...