Accurate replication of the genome requires the evolutionarily conserved minichromosome maintenance protein, Mcm10. Although the details of the precise role of Mcm10 in DNA replication are still debated, it interacts with the Mcm2-7 core helicase, the lagging strand polymerase, DNA polymerase-α and the replication clamp, proliferating cell nuclear antigen. Loss of these interactions caused by the depletion of Mcm10 leads to chromosome breakage and cell cycle checkpoint activation. However, whether Mcm10 has an active role in DNA damage prevention is unknown. Here, we present data that establish a novel role of the N-terminus of Mcm10 in resisting DNA damage. We show that Mcm10 interacts with the Mec3 subunit of the 9-1-1 clamp in response to replication stress evoked by UV irradiation or nucleotide shortage. We map the interaction domain with Mec3 within the N-terminal region of Mcm10 and demonstrate that its truncation causes UV light sensitivity. This sensitivity is not further enhanced by a deletion of MEC3, arguing that MCM10 and MEC3 operate in the same pathway. Since Rad53 phosphorylation in response to UV light appears to be normal in N-terminally truncated mcm10 mutants, we propose that Mcm10 may have a role in replication fork restart or DNA repair.
Minichromosome maintenance protein (Mcm) 10 is a part of the eukaryotic replication machinery and highly conserved throughout evolution. As a multivalent DNA scaffold, Mcm10 coordinates the action of proteins that are indispensable for lagging strand synthesis, such as the replication clamp, proliferating cell nuclear antigen (PCNA). The binding between Mcm10 and PCNA serves an essential function during DNA elongation and is mediated by the ubiquitination of Mcm10. Here we map lysine 372 as the primary attachment site for ubiquitin on S. cerevisiae Mcm10. Moreover, we identify five additional lysines that can be ubiquitinated. Mutation of lysine 372 to arginine ablates ubiquitination of overexpressed protein and causes sensitivity to the replication inhibitor hydroxyurea in cells that are S-phase checkpoint compromised. Together, these findings reveal the high selectivity of the ubiquitination machinery that targets Mcm10 and that ubiquitination has a role in suppressing replication stress.
To ensure genome integrity, cells must replicate with absolute fidelity. However, cells are constantly under stress that causes DNA damage or block replication. To circumvent these problems, cells have a quality control system termed DNA damage checkpoint. In humans, defects in this checkpoint cause genome instability and a strong predisposition to cancer. A recent study has identified minichromosome maintenance protein 10 (Mcm10) as one of the few replication proteins that prevent DNA damage. However, whether Mcm10 has an active role in preventing DNA damage is unknown. We have observed that Mcm10 and mediator of replication checkpoint 1 (Mrc1) interact in S. cerevisiae whole cell extracts. Moreover, a robust interaction was observed between Mcm10 and Mrc1 by yeast two‐hybrid assays. Our data suggests Mcm10 interacts through the conserved C‐terminus of Mrc1. Interestingly, it is the N‐terminus of Mcm10 that binds to Mrc1. We are currently conducting experiments to further elucidate this interaction. These studies promise to identify a novel role of Mcm10 in DNA damage response pathway and maintenance of genomic integrity. Grant Funding Source: Supported by the National Institute of General Medical Sciences of the National Institutes of Health through Grant Number 8P20GM103447 to SDB.
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