Three DNA polymerases, recruited by different mechanisms, carry out NER repair synthesis in human cells

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both polκ and polδ, and both polymerases can be recovered in the same repair complexes. Polκ is recruited to repair sites by ubiquitinated PCNA and XRCC1 and polδ by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on polɛ, recruitment of which is dependent on the alternative clamp loader CTF18-RFC

Transcription-associated breaks in Xeroderma Pigmentosum group D cells from patients with combined features of Xeroderma Pigmentosum and Cockayne Syndrome

Defects in the XPD gene can result in several clinical phenotypes, including xeroderma pigmentosum (XP), trichothiodystrophy, and, less frequently, the combined phenotype of XP and Cockayne syndrome (XP-D/CS). We previously showed that in cells from two XP-D/CS patients, breaks were introduced into cellular DNA on exposure to UV damage, but these breaks were not at the sites of the damage. In the present work, we show that three further XP-D/CS patients show the same peculiar breakage phenomenon. We show that these breaks can be visualized inside the cells by immunofluorescence using antibodies to either gamma-H2AX or poly-ADP-ribose and that they can be generated by the introduction of plasmids harboring methylation or oxidative damage as well as by UV photoproducts. Inhibition of RNA polymerase II transcription by four different inhibitors dramatically reduced the number of UV-induced breaks. Furthermore, the breaks were dependent on the nucleotide excision repair (NER) machinery. These data are consistent with our hypothesis that the NER machinery introduces the breaks at sites of transcription initiation. During transcription in UV-irradiated XP-D/CS cells, phosphorylation of the carboxy-terminal domain of RNA polymerase II occurred normally, but the elongating form of the polymerase remained blocked at lesions and was eventually degraded

Heterochromatin protein 1 is recruited to various types of DNA damage

Heterochromatin protein 1 (HP1) family members are chromatin-associated proteins involved in transcription, replication, and chromatin organization. We show that HP1 isoforms HP1-α, HP1-β, and HP1-γ are recruited to ultraviolet (UV)-induced DNA damage and double-strand breaks (DSBs) in human cells. This response to DNA damage requires the chromo shadow domain of HP1 and is independent of H3K9 trimethylation and proteins that detect UV damage and DSBs. Loss of HP1 results in high sensitivity to UV light and ionizing radiation in the nematode Caenorhabditis elegans, indicating that HP1 proteins are essential components of DNA damage response (DDR) systems. Analysis of single and double HP1 mutants in nematodes suggests that HP1 homologues have both unique and overlapping functions in the DDR. Our results show that HP1 proteins are important for DNA repair and may function to reorganize chromatin in response to damage

Specialized interfaces of Smc5/6 control hinge stability and DNA association

The Structural Maintenance of Chromosomes (SMC) complexes: cohesin, condensin and Smc5/6 are involved in the organization of higher-order chromosome structure—which is essential for accurate chromosome duplication and segregation. Each complex is scaffolded by a specific SMC protein dimer (heterodimer in eukaryotes) held together via their hinge domains. Here we show that the Smc5/6-hinge, like those of cohesin and condensin, also forms a toroidal structure but with distinctive subunit interfaces absent from the other SMC complexes; an unusual ‘molecular latch’ and a functional ‘hub’. Defined mutations in these interfaces cause severe phenotypic effects with sensitivity to DNA-damaging agents in fission yeast and reduced viability in human cells. We show that the Smc5/6-hinge complex binds preferentially to ssDNA and that this interaction is affected by both ‘latch’ and ‘hub’ mutations, suggesting a key role for these unique features in controlling DNA association by the Smc5/6 complex

A novel SMC protein complex in Schizosaccharomyces pombe contains the Rad18 DNA repair protein

In Schizosaccharomyces pombe, rad18 is an essential gene involved in the repair of DNA damage produced by ionizing radiation and in tolerance of UV-induced DNA damage. The Rad18 protein is a member of the SMC (structural maintenance of chromosomes) superfamily, and we show that, like the other SMC proteins in condensin and cohesin, Rad18 is a component of a high-molecular-weight complex. This complex contains at least six other proteins, the largest of which is Spr18, a novel SMC family member closely related to Rad18, and likely to be its heterodimeric partner. SMC proteins have ATP-binding domains at the N- and C-termini, and two extended coiled-coil domains separated by a hinge in the middle. We show that the N-terminal ATP-binding domain of Rad18 is essential for all functions, and overexpression of an N-terminal mutant has a dominant-negative effect. We have identified an important mutation (S1045A) near the C-terminus of Rad18 that separates its repair and essential roles. Potential models for the role of the Rad18¿Spr18 complex during DNA repair are discussed

Xeroderma Pigmentosum

No description supplie

Replication factor C recruits DNA polymerase delta to sites of nucleotide excision repair but is not required for PCNA recruitment

Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre-and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3 termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5 phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA. A multitude of endogenous and exogenous genotoxic agents induce damage to DNA. When not repaired properly, these DNA lesions can interfere with replication and transcription and thereby induce deleterious events (i.e., cell death, mutations, and genomic instability) that affect the fate of organisms (18). To ensure the maintenance of the DNA helix integrity, a network of defense mechanisms has evolved including accurate and efficient DNA repair processes. One of these processes is the nucleotide excision repair (NER) pathway that removes a wide range of DNA helix-distorting lesions, such as sunlightinduced photodimers, for example, cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidone photoproducts (6-4PP). Within NER, more than 30 polypeptides act coordinately, starting from the detection and removal of the lesion up to the restoration of the DNA sequence and chromatin structure. The importance of NER is underlined by the severe clinical consequences associated with inherited NER defects, causing UV-hypersensitive autosomal recessive syndromes: the cancer-predisposing xeroderma pigmentosum (XP) and the premature ageing and neurodegenerative disorders Cockayne syndrome (CS) and trichothiodystrophy (TTD) (27). The initial DNA damage recognition step in NER involves two subpathways: transcription-coupled repair (TCR) and global genome repair (GGR). TCR is responsible for the rapid removal of transcription-blocking DNA lesions and is initiated when elongating RNA polymerase II stalls at a DNA lesion on the transcribed strand (16). In GGR, which removes lesions throughout the genome, damage recognition is facilitated by the concerted action of the heterodimeric XP group C (XPC)-HR23B protein complex and by the UV-damaged DNA-binding protein (UV-DDB) complex (10, 33). Subsequently, the 10-subunit TFIIH complex unwinds the DNA around the lesion. This partially unwound structure is stabilized by the single-strand binding protein replication protein A (RPA) and the damage-verifying protein XPA. Collectively, these proteins load and properly orient the structure-specific endonucleases XPF-ERCC1 and XPG that incise 5Ј and 3Ј of the damage, respectively, creating a single-strand gap of approximately 30 nucleotides (nt

Replication Factor C Recruits DNA Polymerase δ to Sites of Nucleotide Excision Repair but Is Not Required for PCNA Recruitment▿

Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3′ termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5′ phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA