ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage

Abstract

Nuclear Rad51 focus formation is required for homologydirected repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions.

A breathtaking phenotype: Unexpected roles of the DNA base damage response protein ASCIZ as a key regulator of early lung development

“…8 In addition to H 2 O 2 , Asciz-null MEFs were also hypersensitive to MMS but not to other DNA damaging agents, 5,7 corroborating the particular importance of ASCIZ for the survival of BER-processed DNA lesions as expected from the earlier studies in human and chicken cells. [2][3][4] While the two reports are in agreement with regard to the importance of the protein in the response to DNA base damage in vivo, there remains some disagreement about the proposed regulatory role of ASCIZ in ATM activation. In contrast to the earlier claim that ASCIZ was required for ATM stability, 5 ATM levels were unaffected in tissue extracts from our Asciz-null mice, human cells after almost complete siRNA-mediated ASCIZ depletion, as well as in two independently generated chicken Asciz-null cell lines.…”

ATMIN (ATM Interactor)

“…Human and mouse ASCIZ exhibit atypical electrophoretic mobility on SDS-PAGE Western blots with an apparent mass of ~115 kDa rather than the predicted ~88 kDa (McNees et al, 2005). The aberrant mobility is unlikely to be caused by post-translational modifications, as similar mobility retardation is also observed with bacterially expressed recombinant fragments encompassing the SQ/TQ cluster.…”

“…In Northern and Western blot experiments, the two main isoforms of ASCIZ/ATMIN are expressed at relatively similar levels in a wide range of human tissues and all cancer-derived cell lines tested (McNees et al, 2005). Northern blots of various mouse tissues also indicate relatively similar expression levels in a wide range of organs (Jurado et al, 2010).…”

“…The ASCIZ protein is predominantly, if not exclusively, located to the nucleus but not the nucleolus (McNees et al, 2005;Kanu and Behrens, 2007). The protein forms discrete sub-nuclear foci after treatment with methylating and oxidating DNA base damaging agents (McNees et al, 2005;Rapali et al, 2011).…”

“…Protein expression has not been systematically analysed in human tissues but is expected to be similar to mRNA expression profiles by Northern blots suggesting ubiquitous expression (McNees et al, 2005).…”