Phosphorylation-mediated interactions with TOPBP1 couple 53BP1 and 9-1-1 to control the G1 DNA damage checkpoint

Abstract

Coordination of the cellular response to DNA damage is organised by multi-domain ‘scaffold’ proteins, including 53BP1 and TOPBP1, which recognise post-translational modifications such as phosphorylation, methylation and ubiquitylation on other proteins, and are themselves carriers of such regulatory signals. Here we show that the DNA damage checkpoint regulating S-phase entry is controlled by a phosphorylation-dependent interaction of 53BP1 and TOPBP1. BRCT domains of TOPBP1 selectively bind conserved phosphorylation sites in the N-terminus of 53BP1. Mutation of these sites does not affect formation of 53BP1 or ATM foci following DNA damage, but abolishes recruitment of TOPBP1, ATR and CHK1 to 53BP1 damage foci, abrogating cell cycle arrest and permitting progression into S-phase. TOPBP1 interaction with 53BP1 is structurally complimentary to its interaction with RAD9-RAD1-HUS1, allowing these damage recognition factors to bind simultaneously to the same TOPBP1 molecule and cooperate in ATR activation in the G1 DNA damage checkpoint.

Structure-function analysis of TOPBP1’s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system

“…We note that S642 sits within an “SQ” motif, which is a site commonly phosphorylated by ATM and ATR kinases, raising the possibility that phosphorylation of S642 is important for signaling. To date the only known factors that bind TOPBP1’s BRCT4&5 region are the BLM helicase and 53BP1 14 , 36 , 37 , 40 . BLM plays an important role during end resection, and is thus present at DSBs.…”

“…The interaction between RAD9 and TOPBP1 is not required for ATR activation per se, but it is required for ATR to access a subset of its substrates, including CHK1 12 . Other factors also bind TOPBP1 in its BRCT1&2 region, and these include MDC1 and 53BP1 13 , 14 . The interaction with MDC1 bridges TOPBP1 to the MRN complex, as well as the ATM protein kinase, and this is thought to be important for ATR signaling at DSBs 13 , 15 , 16 .…”

“…The interaction with MDC1 bridges TOPBP1 to the MRN complex, as well as the ATM protein kinase, and this is thought to be important for ATR signaling at DSBs 13 , 15 , 16 . The interaction with 53BP1 also appears to be important for ATR signaling, but in a limited manner that is specific for the G1 phase of the cell cycle 14 . Thus while there is a fair amount known about factors binding to TOPBP1’s BRCT1&2 domains during a DSB response, there is comparatively little known about how the remaining 6 BRCT domains are involved, or if they are even needed for ATR signaling at DSBs.…”

The E2F1 transcription factor and RB tumor suppressor moonlight as DNA repair factors

“…This interaction was one of the first examples of a BRCT domain binding to a phosphorylated protein motif, now recognized as a general feature of BRCT domains [ 24 , 25 ]. Other TopBP1 BRCT domains bind to phosphorylated motifs on other DNA damage response factors, including RAD9, 53BP1 and MDC1, which helps to recruit TopBP1 to damaged DNA [ 26 , 27 ]. In turn, TopBP1 oligomerization at sites of DNA damage allows the BRCT6 domain to bind and recruit phosphorylated E2F1 [ 11 , 28 ].…”

The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis

“…1a). TOPBP1 undergoes prominent enrichment at DSB sites throughout the cell cycle by at least four distinct mechanisms that involve phosphorylation-dependent interactions of alternating combinations of its four phospho-binding BRCT domains (BRCT 1, 2, 5 and 7) with the adaptor proteins 53BP1, RAD9, Treacle and MDC1 12, 10,13,14 TOPBP1 interacts with Casein kinase 2 (CK2) phosphorylated MDC1 via its N-terminal BRCT0-2 module. This interaction is not cell cycle regulated but paradoxically, MDC1 recruits TOPBP1 by direct interaction exclusively in mitosis 10 Here we present a possible solution to this conundrum by identifying cancerous inhibitor of protein phosphatase 2A (CIP2A) as a TOPBP1-interacting protein that promotes TOPBP1 recruitment to sites of DSBs specifically in mitosis.…”