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Function of Cdc45 in DNA Replication and in Response to Genotoxic Stress
Author(s) -
DeBrot Amanda,
Lancaster Cynthia,
Bjornsti MaryAnn
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.798.2
Subject(s) - dna supercoil , control of chromosome duplication , biology , dna replication , eukaryotic dna replication , dna damage , microbiology and biotechnology , helicase , dna , replication factor c , origin recognition complex , genetics , topoisomerase , replication protein a , dna binding protein , gene , transcription factor , rna
DNA replication is a highly conserved and tightly regulated process, with a variety of factors acting to ensure faithful duplication and stability of chromosomal DNA. During replication, positive supercoils form ahead of the advancing replication fork. DNA topoisomerase 1 (Top1) acts to prevent DNA damage by relieving the torsional strain created by these supercoils. The camptothecin (CPT) class of chemotherapeutics target Top1, creating a long‐lived DNA‐Top1‐CPT covalent complex, which leads to S‐phase specific DNA lesions and cellular death. Our lab previously performed a genetic screen to isolate mutant proteins that enhanced cell sensitivity to Top1‐mediated DNA damage. One of the mutant proteins identified was Cdc45G510E, which contains a single amino substitution at residue G 510 . Cells expressing this protein were also hypersensitive to other forms of DNA damage and exhibited collapsed replication forks, suggesting a role for Cdc45 in protecting cells from genotoxic stress. Cdc45 is an essential protein found within the CMG ( C dc45‐ M CM‐ G INS) replicative helicase. Until recently, it was considered a scaffolding protein that had only been identified in eukaryotes. However, sequence alignments and structural studies suggest that Cdc45 may in fact be the eukaryotic ortholog of the archaeal RecJ protein. This is due, in large part, to the conservation of residues within DHH (Asp‐His‐His) motifs in the N‐terminus of Cdc45, which act to coordinate a metal ion and are essential for the nuclease activity of various RecJ proteins. The functional similarities between Cdc45 and RecJ identified thus far are the inclusion within the respective replicative helicases and the ability to bind ssDNA. RecJ has also been determined to aid in DNA damage repair, such as homologous recombination and rescue of stalled replication forks. In our mutational analyses of yeast CDC45 , we demonstrated that the conserved DHH residues were also necessary for Cdc45 function in protecting cells from genotoxic stress. Moreover, several of these residues are also required to maintain cell viability in the absence of exogenous DNA damage.