Summary Completion of DNA replication after replication stress depends on PCNA, which undergoes mono-ubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is poly-ubiquitinated to promote recombination dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by poly-ubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.

Summary Cullin Ring Ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. One substrate, NUSAP1, we demonstrate is an SCFCyclin F substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology, and provides a set of proteins likely to be key indicators of cellular physiology.

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify new DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the ALS candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a PARP-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors and >70% of randomly tested transcription factors localized to sites of DNA damage and approximately 90% were PARP-dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding domain-dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP-dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins.

The signal transduction pathway of the DNA damage response (DDR) is activated to maintain genomic integrity following DNA damage. The DDR promotes genomic integrity by regulating a large network of cellular activities that range from DNA replication and repair to transcription, RNA splicing, and metabolism. In this study we define an interaction between the DDR factor NBS1 and TCOF1, a nucleolar protein that regulates ribosomal DNA (rDNA) transcription and is mutated in Treacher Collins syndrome. We show that NBS1 relocalizes to nucleoli after DNA damage in a manner dependent on TCOF1 and on casein kinase II and ATM, which are known to modify TCOF1 by phosphorylation. Moreover, we identify a putative ATM phosphorylation site that is required for NBS1 relocalization to nucleoli in response to DNA damage. Last, we report that TCOF1 promotes cellular resistance to DNA damaging agents. Collectively, our findings identify TCOF1 as a DDR factor that could cooperate with ATM and NBS1 to suppress inappropriate rDNA transcription and maintain genomic integrity after DNA damage.T he faithful conservation of genomic information is an essential process for cell survival and for preventing malignant transformation (1). To maintain genomic integrity, DNA has to be protected from damage either spontaneously induced or generated by environmental sources, including ionizing radiation or chemical agents. The DNA damage response (DDR) is a signal transduction network that is activated to maintain genomic integrity after DNA damage (1, 2). A principal component of the DDR is the ATM kinase, which is primarily activated by the presence of DNA double-strand breaks (DSBs).DSBs are deleterious DNA lesions that can lead to cell death if unresolved. DSBs are fixed either by joining the two DNA ends together by nonhomologous end joining (NHEJ) or by homologydirected repair mediated by homologous recombination (HR) (3). The regulation of DSB end-processing represents a key step in the choice between NHEJ and HR. Whereas NHEJ occurs with minimal end-processing, extensive resection of DNA ends and formation of single-stranded DNA regions is required for the initiation of HR (4, 5).NBS1 is a critical component of the heterotrimeric MRE11-RAD50-NBS1 (MRN) complex, which plays a central role in the repair of DSBs through the activation of the DDR and the initiation of HR. After binding and stabilizing DSB ends, the MRN complex recruits ATM and the mediator protein MDC1 to the break site through their direct interaction with NBS1. MDC1 subsequently associates with the phosphorylated histone variant H2AX (γH2AX) locally to amplify the ATM signaling cascade at DSBs (6-9). Direct interaction with NBS1 also promotes the recruitment of the DNA repair factor CtIP to DSB ends by the MRN complex, where it promotes end resection to initiate HR (10). The importance of NBS1 to the maintenance of genomic integrity is further highlighted by the predisposition to growth defects, craniofacial abnormalities, and B-cell lymphomas of patients with Nijmegen breaka...

Proliferating cell nuclear antigen (PCNA)-associated KIAA0101/PAF15 protein is a cell cycle-regulated anaphase-promoting complex/cyclosome substrate.

DNA lesions can block replication forks and lead to the formation of single-stranded gaps. These replication complications are mitigated by DNA damage tolerance mechanisms, which prevent deleterious outcomes such as cell death, genomic instability, and carcinogenesis. The two main tolerance strategies are translesion DNA synthesis (TLS), in which low-fidelity DNA polymerases bypass the blocking lesion, and homology-dependent repair (HDR; postreplication repair), which is based on the homologous sister chromatid. Here we describe a unique high-resolution method for the simultaneous analysis of TLS and HDR across defined DNA lesions in mammalian genomes. The method is based on insertion of plasmids carrying defined site-specific DNA lesions into mammalian chromosomes, using phage integrase-mediated integration. Using this method we show that mammalian cells use HDR to tolerate DNA damage in their genome. Moreover, analysis of the tolerance of the UV light-induced 6-4 photoproduct, the tobacco smokeinduced benzo[a]pyrene-guanine adduct, and an artificial trimethylene insert shows that each of these three lesions is tolerated by both TLS and HDR. We also determined the specificity of nucleotide insertion opposite these lesions during TLS in human genomes. This unique method will be useful in elucidating the mechanism of DNA damage tolerance in mammalian chromosomes and their connection to pathological processes such as carcinogenesis.error-prone DNA repair | homologous recombination repair | recombinational repair D NA damage is abundant, caused by both external agents such as sunlight and tobacco smoke and intracellular byproducts of metabolism, amounting to about 50,000 lesions per day per cell (1). Despite the presence of effective DNA repair mechanisms that eliminate lesions and restore the original DNA sequence, DNA replication often encounters unrepaired lesions that have escaped repair. These DNA damages may cause arrest of replication forks and the generation of postreplication gaps (2, 3). To complete replication and prevent the formation of double-strand breaks, which are highly deleterious, cells use DNA damage tolerance (DDT) mechanisms. These include translesion DNA synthesis (TLS) and homology-dependent repair (HDR), which enable bypass of the lesions and completion of replication, without removing the lesions from DNA. HDR uses the sequence from the intact sister chromatid to patch the single-stranded template region carrying the lesion. [We term HDR the pathways of DNA damage tolerance that rely on the homologous sister chromatid, also termed postreplication repair (PRR), damage avoidance, template switch, copy choice recombination, and homologous recombination repair.] This is carried out either by physical transfer of the segment complementary to the damaged template [also termed homologous recombination repair (HRR)] or by copying the complementary strand from the sister chromatid (template switch or postreplication repair). TLS employs specialized low-fidelity DNA polymerases to replicate across ...

Tolerance mechanisms are important in the ability of cells to cope with DNA damage. In E. coli, the two main damage tolerance mechanisms are recombinational repair (RR) and translesion replication (TLR). Here we show that RR effectively repairs gaps opposite DNA lesions. When both mechanisms are functional, RR predominates over TLR, being responsible for 86% of the repair events. This predominance of RR is determined by the high concentration of RecA present under SOS conditions, which causes a differential inhibition of TLR. Further inhibition of TLR is caused by the RecA-catalyzed strand exchange reaction of RR. This molecular hierarchy in the tolerance of DNA lesions ensures that the nonmutagenic RR predominates over the mutagenic TLR, thereby contributing to genetic stability.

Damages in the DNA template inhibit the progression of replication, which may cause single-stranded gaps. Such situations can be tolerated by translesion DNA synthesis (TLS), or by homology-dependent repair (HDR), which is based on transfer or copying of the missing information from the replicated sister chromatid. Whereas it is well established that TLS plays an important role in DNA damage tolerance in mammalian cells, it is unknown whether HDR operates in this process. Using a newly developed plasmid-based assay that distinguishes between the three mechanisms of DNA damage tolerance, we found that mammalian cells can efficiently utilize HDR to repair DNA gaps opposite an abasic site or benzo[a]pyrene adduct. The majority of these events occurred by a physical strand transfer (homologous recombination repair; HRR), rather than a template switch mechanism. Furthermore, cells deficient in either the human RAD51 recombination protein or NBS1, but not Rad18, exhibited decreased gap repair through HDR, indicating a role for these proteins in DNA damage tolerance. To our knowledge, this is the first direct evidence of gap-lesion repair via HDR in mammalian cells, providing further molecular insight into the potential activity of HDR in overcoming replication obstacles and maintaining genome stability.