Given its significant role in the maintenance of genomic stability, histone methylation has been postulated to regulate DNA repair. Histone methylation mediates localization of 53BP1 to a DNA double-strand break (DSB) during homologous recombination repair, but a role in DSB repair by nonhomologous end-joining (NHEJ) has not been defined. By screening for histone methylation after DSB induction by ionizing radiation we found that generation of dimethyl histone H3 lysine 36 (H3K36me2) was the major event.Using a novel human cell system that rapidly generates a single defined DSB in the vast majority of cells, we found that the DNA repair protein Metnase (also SETMAR), which has a SET histone methylase domain, localized to an induced DSB and directly mediated the formation of H3K36me2 near the induced DSB. This dimethylation of H3K36 improved the association of early DNA repair components, including NBS1 and Ku70, with the induced DSB, and enhanced DSB repair. In addition, expression of JHDM1a (an H3K36me2 demethylase) or histone H3 in which K36 was mutated to A36 or R36 to prevent H3K36me2 formation decreased the association of early NHEJ repair components with an induced DSB and decreased DSB repair. Thus, these experiments define a histone methylation event that enhances DNA DSB repair by NHEJ.double-strand break | I-Sce-I | chromatin immunoprecipitation | MRN complex | mathematical modeling H istone methylation is highly regulated by a family of proteins termed histone methylases, which usually share a SET domain (1-3). Histone methylation plays a key role in chromatin remodeling and as such regulates transcription, replication, cell differentiation, genome stability, and apoptosis (1-3). Because of its role in replication and genome stability, histone methylation has been hypothesized to play an important role in DNA repair. DNA double-strand breaks (DSBs) are a cytotoxic form of DNA damage that disrupts many of the cellular functions regulated by histone methylation described above (4-6). Previous reports indicate that histone methylation may be important in DNA DSB repair by homologous recombination: The DSB repair component 53BP1, which is required for proper homologous recombination, is recruited to sites of damage by methylated histone H3 lysine 79 (H3K79) and histone H4 lysine 20 (H4K20) (7-9). However, neither H3K79 nor H4K20 methylation is induced by DNA damage (9), so other histone methylation events at sites of DNA damage have been sought. In addition, a mechanism by which histone methylation might regulate NHEJ DSB repair has yet to be defined. In this study, a survey of histone methylation events after DSB induction revealed that the major immediate H3 methylation event is H3K36me2.Metnase is a DNA DSB repair component that is a fusion of a SET histone methylase domain with a nuclease domain and a domain from a member of the transposase/integrase family (10-14). We showed previously that Metnase enhances nonhomologous end-joining (NHEJ) repair of, and survival after, DNA DSBs, and that its SET dom...

Homologous recombinational repair (HRR) of DNA damage is critical for maintaining genome stability and tumor suppression. RAD51 and BRCA2 colocalization in nuclear foci is a hallmark of HRR. BRCA2 has important roles in RAD51 focus formation and HRR of DNA double-strand breaks (DSBs). We previously reported that BCCIP␣ interacts with BRCA2. We show that a second isoform, BCCIP␤, also interacts with BRCA2 and that this interaction occurs in a region shared by BCCIP␣ and BCCIP␤. We further show that chromatin-bound BRCA2 colocalizes with BCCIP nuclear foci and that most radiation-induced RAD51 foci colocalize with BCCIP. Reducing BCCIP␣ by 90% or BCCIP␤ by 50% by RNA interference markedly reduces RAD51 and BRCA2 foci and reduces HRR of DSBs by 20-to 100-fold. Similarly, reducing BRCA2 by 50% reduces RAD51 and BCCIP foci. These data indicate that BCCIP is critical for BRCA2-and RAD51-dependent responses to DNA damage and HRR.DNA double-strand breaks (DSBs) are induced by exogenous agents, such as ionizing radiation (IR), and arise spontaneously during normal DNA metabolism, such as at blocked or collapsed replication forks (9,10,39,45). Defects in DSB repair confer genome instability associated with tumorigenesis. In mammalian cells, DSBs are repaired by nonhomologous end-joining and by homologous recombinational repair (HRR) (60,62,65). RAD51 binds single-stranded DNA (ssDNA) to form nucleoprotein filaments that are essential for strand transfer during HRR (23,44,61,66). RAD51 is normally dispersed in the nucleus, but upon DNA damage induction, it redistributes to nuclear foci that are presumed sites of HRR (6,7,14,20,31,46). RAD51 foci have been shown to be associated with ssDNA regions after DNA damage (46). Several HRR proteins, including XRCC2, XRCC3, RAD51B, RAD51C, RAD51D, and BRCA2, are important for RAD51 focus formation (1,5,7,43,55,56).BRCA2 has nine RAD51 binding regions, including eight BRC repeats encoded by exon 11 and a distinct RAD51 binding region encoded by exon 27 (8, 33, 69). Expression of individual BRC repeats interferes with RAD51 focus formation and HRR (5, 53, 70), indicating that RAD51-BRCA2 interactions are important for both processes. The C-terminal half of BRCA2 has three regions that are structurally related to the ssDNA binding region of RPA and bind ssDNA in vitro, suggesting that ssDNA binding is also important for BRCA2 function in HRR (71). These ssDNA binding regions occur in a region called conserved domain IV (30,48,73) or the BRCA2 C-terminal domain (71), which is the longest and most evolutionarily conserved BRCA2 domain (32, 57). This domain also has binding sites for several proteins including DSS1, BUBR1, ABP-280/filamin-A, and BCCIP␣ (16,30,34,73).BCCIP␣ is a BRCA2 and CDKN1A (p21, Cip1, and Waf1) interaction protein (30); it has also been called . A second isoform, BCCIP␤, shares an N-terminal acidic domain and a central conserved domain but has a distinct C-terminal domain (Fig. 1A). In this report, BCCIP indicates both proteins. The BCCIP proteins share no significan...

Metnase is a human SET and transposase domain protein that methylates histone H3 and promotes DNA double-strand break repair. We now show that Metnase physically interacts and co-localizes with Topoisomerase IIα (Topo IIα), the key chromosome decatenating enzyme. Metnase promotes progression through decatenation and increases resistance to the Topo IIα inhibitors ICRF-193 and VP-16. Purified Metnase greatly enhanced Topo IIα decatenation of kinetoplast DNA to relaxed circular forms. Nuclear extracts containing Metnase decatenated kDNA more rapidly than those without Metnase, and neutralizing anti-sera against Metnase reversed that enhancement of decatenation. Metnase automethylates at K485, and the presence of a methyl donor blocked the enhancement of Topo IIα decatenation by Metnase, implying an internal regulatory inhibition. Thus, Metnase enhances Topo IIα decatenation, and this activity is repressed by automethylation. These results suggest that cancer cells could subvert Metnase to mediate clinically relevant resistance to Topo IIα inhibitors.