DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination

Abstract

Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome.NA double strand breaks (DSBs) are a most severe type of DNA damage caused by endogenous metabolic processes and exogenous exposure to radiation and chemicals. Unrepaired DSBs induce genomic instability, carcinogenesis, and premature aging. In mammalian cells, DSBs are repaired by either the nonhomologous end joining (NHEJ) or the homologous recombination (HR) pathway. Although it is a common understanding that HR primarily takes place in response to strand breaks in the S-G2 phases of the cell cycle where the undamaged sister chromatids are present as donor templates, recent studies have suggested that homologous pairing also occurs during the G0/G1 phase and is associated with transcription (1), although the mechanisms remain to be elucidated. At active transcription sites, RNA polymerase II (RNA POLII) can bypass base modifications such as 8-oxo guanine but not single strand breaks (SSBs) and DSBs (2-5), indicating that unrepaired strand breaks at transcriptionally active (TA) sites can be especially deleterious and may lead to secondary damage.The Cockayne syndrome B (CSB) gene is defective in approximately two-thirds of patients with Cockayne syndrome (CS), an autosomal recessive disease with diverse clinical signs including severe growth failure, progressive neurodegeneration, and hypersensitivity to sunlight. CSB has an established role in transcription-coupled nucleotide excision repair (TC-NER) of photo lesions. When RNA POLII is stalled at bulky lesions, CSB is loaded to facilitate NER of the transcribed strand (6, 7). As noted, in addition to UV sensitivity, CS patients also manifest severe neurodegeneration (8, 9), suggesting the importance of CS proteins in maintaining genome stability against a broad spectrum of DNA damage. For example, CSB-defective cells are also sensitive to ionizing radiation (IR) (10, 11), which is phenotypically distinctive from classic NER deficiencies and indicates that CSB function is not limited to UV-derived photo lesions. In addition, CSB-deficient mice exhibit a subset of sy...

Elements That Regulate the DNA Damage Response of Proteins Defective in Cockayne Syndrome

“…We also found that mutation of an essential residue for the ATPase activity of CSB (K538A) did not alter the recruitment of the protein to oxidative DNA lesions, angelicin monoadducts or trioxsalen ICLs, indicating that targeted accumulation of CSB does not require enzymatic activity. Our finding is generally consistent with the observation that a CSB ATPase mutant protein detectably localized to sites of DSBs 29 and associated with chromatin normally following treatment of cells with the genotoxin etoposide 30 . Moreover, prior reconstitution studies found that assembly of the TC-NER complex, involving proteins such as TFIIH, XPA, RPA, XPG, XPF and CSB, was independent of ATP 59 .…”

“…Consistent with ICLs being recognized, at least in part, via a transcription-associated repair mechanism, we found that α-amanitin, an RNAPII inhibitor, significantly reduced CSB accumulation at trioxsalen DNA crosslinks 24 . Recent studies have also indicated that DSBs may be processed in a CSB-facilitated mechanism that involves active transcription 29 . We report herein that RNAPII inhibition similarly impairs CSB recruitment to angelicin monoadducts, a phenomenon that might be predicted for these bulky modifications, and is generally consistent with prior experiments demonstrating that α-amanitin reduces TC-NER protein (e.g., CSB and SMARCA5) accumulation at UV photoproducts 55 .…”

“…Indeed, a CSB ATPase mutant protein poorly complements the UV light sensitivity of CS cells, indicating that this functionality plays a pivotal role in DNA damage removal 43, 60 . Furthermore, the ATPase activity of CSB has been reported to be vital for the assembly of NER or homologous recombination proteins needed to execute resolution of DNA photoproducts or DSBs 29, 45 . That said, additional work has suggested that, while the ATPase activity of CSB may be important to TC-NER and DSB repair, this enzymatic function does not influence the processing of the oxidative base lesion 8-oxoguanine 61 .…”

“…Moreover, our data indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving an interaction with the 5′-3′ exonuclease SNM1A 24 . Finally, it has recently been shown that CSB modulates the resolution of DSBs via homologous recombination, possibly in a transcription-associated process that engages RAD51C and RAD52, and checkpoint activation 29, 30 . Studies suggest that the accumulation of DNA damage in CS cells may promote mitochondrial dysfunction via a hyperactive PARP1 response 31 .…”

DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

“…However, recent evidence suggests that novel DSB repair pathways involving RNA templates may be functional throughout the cell cycle, including G 1 . RNA template-guided DSB repair has been reported in both mammalian cells and bacteria and involves RNA molecules that serve as templates to guide DNA synthesis during DSB repair (72)(73)(74)(75)(76). Regulation of RNA-DNA duplexes, including both their formation and resolution, may be critical to this repair process.…”