Role of TopBP1-Msh2 interaction in ATR-Chk1 Pathway

Abstract

The cell cycle checkpoint signaling cascade maintains integrity of the eukaryotic genome. It is activated upon DNA damage and it responds to a variety of genetic lesions like single or double strand breaks and damage caused by UV or chemicals. Two canonical DNA damage signaling pathways operate namely the ATR (Ataxia Telangiectasia Rad3 related) and ATM (Ataxia Telangiectasia mutated) pathways. In these pathways the key sensor kinases detect stalled replication forks or strand breaks and signal the effector kinases Chk1 or Chk2 respectively, thus delaying the cell cycle until the DNA is repaired. DNA damage initiates a signaling cascade that involves a number of protein interactions. We focused on the interaction between Topoisomerase IIβ-binding protein (TopBP1) and MutS homolog 2 (Msh2). Though TopBP1 was identified earlier as a binding partner of Msh2, the functional relevance of this interaction is still not well understood. TopBP1 is an important scaffolding protein that detects single stranded breaks and activates ATR kinase upon DNA damage. Human TopBP1 protein contains nine BRCT domains that are capable of multiple protein-protein interactions, which in turn regulate processes like DNA replication initiation and checkpoint activation. Msh2 is a key player in the mismatch repair (MMR) signaling and is commonly mutated in colorectal cancers (HNPCCs). Msh2 along with Msh6 scan the DNA in search of any mismatch that may have occurred during replication. Msh2 has recently been implicated in checkpoint activation following methylation damage and has been shown to interact with ATR following Cisplatin as well as N-Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG) damage. In the current study we elucidate the functional relevance of TopBP1-Msh2 interaction in the ATR-Chk1 pathway following methylation damage. Employing in vitro studies we show that BRCT domains 7 and 8 of TopBP1 interact with the Mutsd and Mutsac domain of Msh2. Our studies show that TopBP1 and Msh2–Msh6 complex exists in vivo and is possibly recruited on the chromatin upon DNA damage. TopBP1 formed damage-induced nuclear foci following N-methyl-N-nitrosourea (MNU) damage while Msh2 only translocated to the nucleus. The accumulation of Msh2 in the nucleus was found to increase over time. Knockdown studies show that TopBP1 is essential for Chk1 activation (phosphorylation at Ser317 and Ser345). On the other hand when we knockdown Msh2, Chk1 phosphorylation by ATR kinase was limited to Ser345. Previous reports have claimed that phosphorylation at Ser 317 of Chk1 to be a priming site for complete activation of Chk1 following DNA damage. However our observation shows that phosphorylation of Chk1 at Ser 345 may be independent of the priming effect of S317 in Msh2 null background. Our studies point towards the role of a checkpoint protein, TopBP1 and a mismatch repair protein, Msh2 to be involved in regulation of DNA damage signaling. As the complex between MutS and TopBP1 is constitutively associated, it is quite possible that the complex could be scanning DNA for any kind of damage irrespective of cell cycle phase. The partial disruption of the checkpoint signaling following Msh2 knockdown suggests that there might exist a separate mechanism of phosphorylation of Chk1 at Ser 317, which may or may not be dependent on TopBP1-Msh2 interaction. The study provides new insights into the complex regulation of checkpoint signaling where multiple protein complexes are required for every step in the signaling cascade. The study gives a platform to further explore the recruitment of TopBP1-Msh2 complex and also further characterize the substrate specificity of Msh2 towards activation of Chk1 following methylation damage.