C hronic stalling of DNA replication forks by DNA damage such as UV irradiation, ionizing irradiation, chemicals, and reactive cellular metabolites impedes the progression of the cell cycle and eventually causes cell death. To circumvent such situations, cells have evolved the postreplication repair (PRR) pathway that bypasses DNA lesions to resolve stalled forks without removing the actual damage (1). In budding yeast Saccharomyces cerevisiae, PRR is carried out by 2 distinct pathways: translesion synthesis (TLS) and template switching (TS) (Fig. 1A). TLS uses multiple low-fidelity TLS polymerases to incorporate nucleotides across DNA lesions (2, 3). Switching from replicative polymerases ␦ or to TLS polymerases is promoted through the interaction between monoubiquitinated PCNA at lysine 164 (K164) and a ubiquitin-binding motif in TLS polymerases-a mechanism conserved from the budding yeast to human. The monoubiquitination of PCNA at K164 requires the RING-type ubiquitin ligase Rad18 (E3) and the ubiquitinconjugating enzyme Rad6 (E2).The TS pathway bypasses DNA damage by switching a stalled replicating end to the nascent daughter strand of the sister chromatid (1, 4). This pathway involves a lysine 63 (K63)-linked polyubiquitin chain that is further added onto the monoubiquitinated PCNA by Rad5 (E3) along with the Ubc13-Mms2 (E2 and E2 variant, respectively) heterodimer complex (Fig. 1 A). Distinct from the K48-linked polyubiquitination leading to protein degradation, the K63-linked polyubiquitination of PCNA is thought to promote TS in a proteasome-independent manner (5).The importance of the TLS pathway in the suppression of mammalian tumorigenesis emerged with the identification of a mutation in TLS polymerase in patients with the variant form of xeroderma pigmentosum and from studies with mouse models (6, 7). Despite the presence of UBC13 and MMS2 homologues in humans, the importance of the TS pathway is less clear in mammals because K63-linked polyubiquitination of PCNA, a hallmark event for the TS pathway, had not been observed until recently (8-10). We recently identified human SHPRH, which possesses SWI2/SNF2 and RING domains with similar architecture to the yeast Rad5 as a functional homologue of yeast Rad5 (9). Specifically, we demonstrated the in vivo activity of SHPRH in promoting a K63-linked polyubiquitination of PCNA as well as physical interactions of SHPRH with PCNA, RAD18, and UBC13. Depletion of SHPRH increases genomic instability after genotoxic stress. Consistent with our work, another study also demonstrated that SHPRH could polyubiquitinate PCNA in vitro (11).In the present study, we demonstrated that ectopic expression of HLTF/SMARCA3/RUSH/HIP116/Zbu1 (hereafter, HLTF) enhanced PCNA polyubiquitination in vivo. Depletion of SHPRH or HLTF significantly reduced polyubiquitination of chromatin-bound PCNA upon treatment of cells with DNA-damaging agents that cause stalled DNA replication forks. Furthermore, Hltf-deficient mouse embryonic fibroblasts (MEFs) showed elevated chromosome breaks an...

Abnormally methylated genes are increasingly being used as cancer biomarkers 1, 2 . For clinical applications, it is important to precisely determine the number of methylated molecules in the analyzed sample. We here describe a digital approach that can enumerate one methylated molecule out of ~5000 unmethylated molecules. Individual DNA fragments can be amplified and analyzed either by flow cytometry or next generation sequencing instruments. Using methylated vimentin as a biomarker, we tested 191 plasma samples and detected cancer cases with 59% sensitivity (95% CI, 48%-70%) and 93% specificity (95% CI, 86%-97%). Using the same assay, we analyzed 80 stool samples and demonstrated 45% sensitivity for detecting colorectal adenomas (23%-68%), 41% COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturebiotechnology/. NIH Public Access Author ManuscriptNat Biotechnol. Author manuscript; available in PMC 2010 March 30. Published in final edited form as:Nat Biotechnol. 2009 September ; 27(9): 858-863. doi:10.1038/nbt.1559. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript sensitivity for detecting cancer (21%-64%), and 95% specificity (82%-99%). This digital quantification of rare methylation events should be applicable to diagnostic evaluations of clinical samples, to preclinical assessments of new epigenetic biomarkers, and to quantitative analyses of epigenetic biology.In humans, DNA methylation is largely restricted to cytosines within 5′-CpG dinucleotides. This covalent modification of DNA functions as an important mediator of gene regulation and, together with covalent modifications of histone proteins, forms the cornerstone for the burgeoning field of epigenetics. Though cancers are globally hypomethylated 3 , specific regions of genes have been shown to be hypermethylated in association with transcription silencing 4 . In addition to its implications for gene regulation, DNA methylation is providing a new generation of cancer biomarkers 5 . Though mutant sequences provide exquisitely specific biomarkers of this class 6, 7 , their utility is compromised by their heterogeneity: the same gene can be mutationally inactivated through many different mechanisms or mutated at many different positions. In contrast, DNA hypermethylation in cancers often affects identical residues in the regulatory regions of particular genes, providing major advantages in biomarker test design. Accordingly, many studies have employed DNA methylation of specific genes for diagnostics development 2,4,5,8 . Such diagnostic tests can in principle be used for early detection of cancers, for assessing prognosis, and for determining the effects of therapy or detecting residual disease.The majority of diagnostic tests based on DNA methylation have employed bisulfite to convert cytosine residues to uracils. This conversion alters the sequence of DNA 9 , providing an opportunity to assess DNA methylat...

Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63–linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)–induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress.