Ildiko Unk

Physical and Functional Interactions of Human DNA Polymerase η with PCNA

ABSTRACT Human DNA polymerase η (hPolη) functions in the error-free replication of UV-damaged DNA, and mutations in hPolη 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 hPolη is targeted to the replication machinery stalled at a lesion site remains unknown. Here, we provide evidence for the physical interaction of hPolη with proliferating cell nuclear antigen (PCNA) and show that mutations in the PCNA binding motif of hPolη inactivate this interaction. PCNA, together with replication factor C and replication protein A, stimulates the DNA synthetic activity of hPolη, 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 apparentK m for the incoming nucleotide.

Yeast Rad5 Protein Required for Postreplication Repair Has a DNA Helicase Activity Specific for Replication Fork Regression

Summary Lesions 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.

Targeting of human DNA polymerase ι to the replication machinery via interaction with PCNA

Human DNA polymerase ι (hPolι) promotes translesion synthesis by inserting nucleotides opposite highly distorting or noninstructional DNA lesions. Here, we provide evidence for the physical interaction of hPolι with proliferating cell nuclear antigen (PCNA), and show that PCNA, together with replication factor C (RFC) and replication protein A (RPA), stimulates the DNA synthetic activity of hPolι. In the presence of these protein factors, on undamaged DNA, the efficiency (Vmax/Km) of correct nucleotide incorporation by hPolι is increased ≈80–150-fold, and this increase in efficiency results from a reduction in the apparent Km for the nucleotide. PCNA, RFC, and RPA also stimulate nucleotide incorporation opposite the 3′-T of the (6) thymine–thymine (T-T) photoproduct and opposite an abasic site. The interaction of hPolι with PCNA implies that the targeting of this polymerase to the replication machinery stalled at a lesion site is achieved via this association.

Interaction with PCNA Is Essential for Yeast DNA Polymerase η Function

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.

Stimulation of DNA Synthesis Activity of Human DNA Polymerase κ by PCNA

ABSTRACT Humans have three DNA polymerases, Polη, Polκ, and Polι, which are able to promote replication through DNA lesions. However, the mechanism by which these DNA polymerases are targeted to the replication machinery stalled at a lesion site has remained unknown. Here, we provide evidence for the physical interaction of human Polκ (hPolκ) with proliferating cell nuclear antigen (PCNA) and show that PCNA, replication factor C (RFC), and replication protein A (RPA) act cooperatively to stimulate the DNA synthesis activity of hPolκ. The processivity of hPolκ, however, is not significantly increased in the presence of these protein factors. The efficiency (V max/K m ) of correct nucleotide incorporation by hPolκ is enhanced ∼50- to 200-fold in the presence of PCNA, RFC, and RPA, and this increase in efficiency is achieved by a reduction in the apparent K m for the nucleotide. Although in the presence of these protein factors, the efficiency of the insertion of an A nucleotide opposite an abasic site is increased ∼40-fold, this reaction still remains quite inefficient; thus, it is unlikely that hPolκ would bypass an abasic site by inserting a nucleotide opposite the site.

Human SHPRH is a ubiquitin ligase for Mms2–Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen

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 SWI/SNF family of ATPases/helicases, and it possesses a C3HC4 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.

Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination.

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 rad5Δ 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 cancer-suppression role.

Role of yeast Rad5 and its human orthologs, HLTF and SHPRH in DNA damage tolerance

In the yeast Saccharomyces cerevisiae, the Rad6-Rad18 DNA damage tolerance pathway constitutes a major defense system against replication fork blocking DNA lesions. The Rad6-Rad18 ubiquitin-conjugating/ligase complex governs error-free and error-prone translesion synthesis by specialized DNA polymerases, as well as an error-free Rad5-dependent postreplicative repair pathway. For facilitating replication through DNA lesions, translesion synthesis polymerases copy directly from the damaged template, while the Rad5-dependent damage tolerance pathway obtains information from the newly synthesized strand of the undamaged sister duplex. Although genetic data demonstrate the importance of the Rad5-dependent pathway in tolerating DNA damages, there has been little understanding of its mechanism. Also, the conservation of the yeast Rad5-dependent pathway in higher order eukaryotic cells remained uncertain for a long time. Here we summarize findings published in recent years regarding the role of Rad5 in promoting error-free replication of damaged DNA, and we also discuss results obtained with its human orthologs, HLTF and SHPRH.

Role of Double-Stranded DNA Translocase Activity of Human HLTF in Replication of Damaged DNA

ABSTRACT 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.

Roles of PCNA-binding and ubiquitin-binding domains in human DNA polymerase η in translesion DNA synthesis

Treatment of yeast and human cells with DNA-damaging agents elicits Rad6–Rad18-mediated monoubiquitination of proliferating cell nuclear antigen (PCNA) at its Lys-164 residue [ubiquitin (Ub)-PCNA], and this PCNA modification is indispensable for promoting the access of translesion synthesis (TLS) polymerases (Pols) to PCNA. However, the means by which K164-linked Ub modulates the proficiency of TLS Pols to bind PCNA and take over synthesis from the replicative Pol has remained unclear. One model that has gained considerable credence is that the TLS Pols bind PCNA at 2 sites, to the interdomain connector loop via their PCNA-interacting protein (PIP) domain and to the K164-linked Ub moiety via their Ub-binding domain (UBD). Specifically, this model postulates that the UBD-mediated binding of TLS Pols to the Ub moiety on PCNA is necessary for TLS. To test the validity of this model, we examine the contributions that the PIP and Ub-binding zinc finger (UBZ) domains of human Polη make to its functional interaction with PCNA, its colocalization with PCNA in replication foci, and its role in TLS in vivo. We conclude from these studies that the binding to PCNA via its PIP domain is a prerequisite for Polηs ability to function in TLS in human cells and that the direct binding of the Ub moiety on PCNA via its UBZ domain is not required. We discuss the possible role of the Ub moiety on PCNA in TLS.