Supplementary MaterialsDocument S1

Supplementary MaterialsDocument S1. avoid genome instability, cell death, or malignancy (Jackson and Bartek, 2009). Interphase cells respond to DSBs by triggering a signaling cascade to activate cell-cycle checkpoints and DNA repair. In contrast, in mitotic cells there is no DNA damage?checkpoint after MRK-016 prophase (Rieder and Cole, 1998), and DSBs?are transmitted into the following G1 phase for repair to?avoid chromosomal instability (Lee et?al., 2014, Orthwein et?al., 2014). The cellular response to DSBs is usually regulated by three related protein kinases, ataxia-telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) (Blackford and Jackson, 2017). Upon DNA damage, one Mouse monoclonal to GATA1 of the earliest substrates of these kinases is the histone variant H2AX, which is phosphorylated at DSB sites on Ser139 and MRK-016 then referred to as H2AX (Rogakou et?al., 1999). H2AX is usually recognized by MDC1 (Stucki et?al., 2005), a scaffold protein that functions as a platform for recruitment of various DNA damage response factors to mediate DNA repair. One of these is the MRE11-RAD50-NBS1 (MRN) complex, which binds to MDC1 via a direct interaction between the NBS1 subunit of MRN and multiple acidic sequence motifs near the N terminus of MDC1 (Chapman and Jackson, 2008, Hari et?al., 2010, Melander et?al., 2008, Spycher et?al., 2008, Wu et?al., 2008). Another is usually RNF8, an E3 ubiquitin ligase with an FHA domain name that binds to a cluster of conserved threonine residues in MDC1 that are phosphorylated by ATM in response to DSBs to promote chromatin ubiquitylation events required for recruitment of DNA damage response mediator proteins such as 53BP1 and BRCA1 (Huen et?al., 2007, Kolas et?al., 2007, Mailand et?al., 2007). Recruitment of these factors to chromatin-flanking DSB sites channels DNA repair into either the non-homologous end-joining pathway or homology-directed repair via mechanisms that are still not completely comprehended (Hustedt and Durocher, 2016). H2AX and MDC1 form foci at DSBs throughout the cell cycle, but recruitment of downstream factors such as RNF8 and 53BP1 is usually blocked during mitosis (Giunta et?al., 2010, Nakamura et?al., 2010, Nelson et?al., 2009, van Vugt et?al., 2010, Lee et?al., 2014, Orthwein et?al., 2014). However, given that inhibition of ATM and DNA-PK activity in mitosis causes radiosensitivity, it is possible that DNA damage signaling as well as recruitment of MDC1 and potentially some of its downstream factors, play an as-yet unidentified role in dealing with DNA damage in this cell-cycle MRK-016 phase. Here, we identify two highly conserved motifs in MDC1 and show that they are phosphorylated by casein kinase 2 (CK2). We identify the DNA damage response mediator proteins TOPBP1 because the binding partner for these motifs and demonstrate the MRK-016 fact that MDC1-TOPBP1 interaction is certainly specifically necessary for TOPBP1 recruitment to DSBs in mitosis. Lack of MDC1-TOPBP1 binding results in radiosensitivity in mitotic cells, in addition to increased micronuclei development, chromosome/chromatid breaks, and chromosome end-to-end fusions. Outcomes A Conserved Acidic Series Motif close to the N Terminus of MDC1 Binds to TOPBP1 Previously, we among others discovered six conserved acidic series motifs close to the N terminus of MDC1 that straight connect to NBS1 and so are necessary for MRN foci development at sites of DSBs (Chapman and Jackson, 2008, Melander et?al., 2008, Spycher et?al., 2008, Wu et?al., 2008). These motifs include Ser-Asp-Thr residues inserted within an acidic series environment; hence, they are called SDT motifs (Body?1A). The serine and threonine residues inside the SDT motifs are phosphorylated by CK2 constitutively, and the initial bivalent N-terminal FHA/BRCT2 phospho-binding module of NBS1 particularly recognizes and binds to the doubly phosphorylated MDC1 SDT motifs (Hari et?al., 2010, Lloyd et?al., 2009). Open in a separate window Number?1 A Conserved Acidic Sequence Motif near the N Terminus of MDC1 Binds to.