No abstract

Chromatin reorganization plays an important role in DNA repair, apoptosis, and cell cycle checkpoints. Among proteins involved in chromatin reorganization, TIP60 histone acetyltransferase has been shown to play a role in DNA repair and apoptosis. However, how TIP60 regulates chromatin reorganization in the response of human cells to DNA damage is largely unknown. Here, we show that ionizing irradiation induces TIP60 acetylation of histone H2AX, a variant form of H2A known to be phosphorylated following DNA damage. Furthermore, TIP60 regulates the ubiquitination of H2AX via the ubiquitin-conjugating enzyme UBC13, which is induced by DNA damage. This ubiquitination of H2AX requires its prior acetylation. We also demonstrate that acetylation-dependent ubiquitination by the TIP60-UBC13 complex leads to the release of H2AX from damaged chromatin. We conclude that the sequential acetylation and ubiquitination of H2AX by TIP60-UBC13 promote enhanced histone dynamics, which in turn stimulate a DNA damage response.Chromatin reorganization by histone modification and mobilization plays a crucial role in DNA metabolism, including replication, transcription, and repair. It appears that histone modification and mobilization can reorganize chromatin to allow DNA repair machinery to access damaged chromosomal DNA (11,29,52,56,57).H2AX is a histone variant that differs from H2A at various amino acid residues along the entire protein and in its Cterminal extensions. H2AX is phosphorylated after the induction of DNA double-strand breaks (DSBs), and the phosphorylated H2AX (␥-H2AX) participates in focus formation at sites of DNA damage. After induction of DSBs, the MRN complex (MRE11, RAD50, and NBS1) binds to broken DNA ends and recruits active ATM, ATR, and/or DNA protein kinase, resulting in the initial phosphorylation of H2AX (32,38,40). MDC1 then associates with ␥-H2AX and recruits additional activated ATM to the sites of DSBs (23,46). This positive feedback loop leads to the expansion of the ␥-H2AX region surrounding DSBs and provides docking sites for many DNA damage and repair proteins, including the MRN complex, 53BP1, and BRCA1 (5, 6, 46). ␥-H2AX plays a role in the accumulation but not in the initial recruitment of repair factors such as the MRN complex, 53BP1, and BRCA1 (10, 63). Therefore, modifications of H2AX other than phosphorylation could play a role in the initial step of the DNA damage response.Until recently, the biological significance of ubiquitination in the DNA damage response has been unclear. H2B ubiquitination regulates the damage checkpoint response (15). H2A is ubiquitinated during the response to UV-induced DNA damage (8). UV-induced DNA damage also causes the ubiquitination of histones H3 and H4, resulting in their release from chromatin (60). Interestingly, ubiquitin-conjugated proteins appear to be accumulated at sites of DSBs, forming nuclear foci like ␥-H2AX (34). These findings raise the possibility that histone ubiquitination is also involved in the reorganization of chromatin in response to D...

Cells often utilize multiple pathways to repair the same DNA lesion, and pathway choice has profound implications for the fidelity of genome maintenance. DNA interstrand cross-links (ICLs) block DNA replication and transcription by covalently linking the two strands of DNA, and the cytotoxicity of ICLs is exploited for chemotherapy. In Xenopus egg extracts, replication fork collision with ICLs initiates two distinct repair pathways. The NEIL3 glycosylase can cleave the cross-link 1 , but if this fails, the Fanconi anemia (FA) proteins incise the phosphodiester backbone surrounding the ICL, generating a double-strand break (DSB) intermediate that is repaired by homologous recombination 2 . How the simpler NEIL3 pathway is prioritized over the FA pathway, which can cause genomic rearrangements, is unknown. Here, we show that the E3 ubiquitin ligase Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

SUMMARYDNA interstrand crosslinks (ICLs) are extremely cytotoxic, but the mechanism of their repair remains incompletely understood. Using Xenopus egg extracts, we previously showed that repair of a cisplatin ICL is triggered when two replication forks converge on the lesion. After CDC45/MCM2-7/GINS (CMG) ubiquitylation and unloading by the p97 segregase, FANCI-FANCD2 promotes DNA incisions by XPF-ERCC1, leading to ICL unhooking. Here, we report that, during this cell-free ICL repair reaction, one of the two converged forks undergoes reversal. Fork reversal fails when CMG unloading is inhibited, but it does not require FANCI-FANCD2. After one fork has undergone reversal, the opposing fork that still abuts the ICL undergoes incisions. Our data show that replication fork reversal at an ICL requires replisome disassembly. We present a revised model of ICL repair that involves a reversed fork intermediate.

Here we describe the use of reverse-phase liquid chromatography mass spectrometry (RP-LC-MS) to simultaneously characterize variants and post-translationally modified isoforms for each histone. The analysis of intact proteins significantly reduces the time of sample preparation and simplifies data interpretation. LC-MS analysis and peptide mass mapping have previously been applied to identify histone proteins and to characterize their post-translational modifications. However, these studies provided limited characterization of both linker histones and core histones. The current LC-MS analysis allows for the simultaneous observation of all histone PTMs and variants (both replacement and bulk histones) without further enrichment, which will be valuable in comparative studies. Protein identities were verified by the analysis of histone H2A species using RPLC fractionation, AU-PAGE separation and nano-LC-MS/MS.

Background: RAD51 and RecA homologs form a nucleoprotein filament (NPF) that includes an ATP cap, which sandwiches the adenosine nucleotide. Results: The HsRAD51(D316K) ATP cap substitution dramatically enhances recombinase and NPF stability. Conclusion: The HsRAD51(Asp-316) residue forms a salt bridge with ATP that allows more rapid protein turnover. Significance: HsRAD51(D316K) provides a useful reagent for the study of recombinase function in physiological conditions.

High fidelity homologous DNA recombination depends on mismatch repair (MMR), which antagonizes recombination between divergent sequences by rejecting heteroduplex DNA containing excessive nucleotide mismatches. The hMSH2-hMSH6 heterodimer is the first responder in postreplicative MMR and also plays a prominent role in heteroduplex rejection. Whether a similar molecular mechanism underlies its function in these two processes remains enigmatic. We have determined that hMSH2-hMSH6 efficiently recognizes mismatches within a D-loop recombination initiation intermediate. Mismatch recognition by hMSH2-hMSH6 is not abrogated by human replication protein A (HsRPA) bound to the displaced single-stranded DNA (ssDNA) or by HsRAD51. In addition, ATP-bound hMSH2-hMSH6 sliding clamps that are essential for downstream MMR processes are formed and constrained within the heteroduplex region of the D-loop. Moreover, the hMSH2-hMSH6 sliding clamps are stabilized on the Dloop by HsRPA bound to the displaced ssDNA. Our findings reveal similarities and differences in hMSH2-hMSH6 mismatch recognition and sliding-clamp formation between a D-loop recombination intermediate and linear duplex DNA.

Double-strand breaks (DSB) occur in chromatin following replication fork collapse and chemical or physical damage [Symington and Gautier (Double-strand break end resection and repair pathway choice. Annu. Rev. Genet. 2011;45:247–271.)] and may be repaired by homologous recombination (HR) and non-homologous end-joining. Nucleosomes are the fundamental units of chromatin and must be remodeled during DSB repair by HR [Andrews and Luger (Nucleosome structure(s) and stability: variations on a theme. Annu. Rev. Biophys. 2011;40:99–117.)]. Physical initiation of HR requires RAD51, which forms a nucleoprotein filament (NPF) that catalyzes homologous pairing and strand exchange (recombinase) between DNAs that ultimately bridges the DSB gap [San Filippo, Sung and Klein. (Mechanism of eukaryotic HR. Annu. Rev. Biochem. 2008;77:229–257.)]. RAD51 forms an NPF on single-stranded DNA and double-stranded DNA (dsDNA). Although the single-stranded DNA NPF is essential for recombinase initiation, the role of the dsDNA NPF is less clear. Here, we demonstrate that the human RAD51 (HsRAD51) dsDNA NPF disassembles nucleosomes by unwrapping the DNA from the core histones. HsRAD51 that has been constitutively or biochemically activated for recombinase functions displays significantly reduced nucleosome disassembly activity. These results suggest that HsRAD51 can perform ATP hydrolysis-dependent nucleosome disassembly in addition to its recombinase functions.