title: Concerted and differential actions of two enzymatic domains underlie Rad5 contributions to DNA damage tolerance.
abstract: Many genome maintenance factors have multiple enzymatic activities. In most cases, how their distinct activities functionally relate with each other is unclear. Here we examined the conserved budding yeast Rad5 protein that has both ubiquitin ligase and DNA helicase activities. The Rad5 ubiquitin ligase activity mediates PCNA poly-ubiquitination and subsequently recombination-based DNA lesion tolerance. Interestingly, the ligase domain is embedded in a larger helicase domain comprising seven consensus motifs. How features of the helicase domain influence ligase function is controversial. To clarify this issue, we use genetic, 2D gel and biochemical analyses and show that a Rad5 helicase motif important for ATP binding is also required for PCNA poly-ubiquitination and recombination-based lesion tolerance. We determine that this requirement is due to a previously unrecognized contribution of the motif to the PCNA and ubiquitination enzyme interaction, and not due to its canonical role in supporting helicase activity. We further show that Rad5's helicase-mediated contribution to replication stress survival is separable from recombination. These findings delineate how two Rad5 enzymatic domains concertedly influence PCNA modification, and unveil their discrete contributions to stress tolerance.
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA Programs in Biochemistry, Cell, and Molecular Biology, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021, USA.
authors.forename: Koyi
authors.initials: K
authors.lastname: Choi
authors.affiliation: Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Batke
authors.affiliation: IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Via Adamello 16, 20139, Italy.
authors.forename: Barnabas
authors.initials: B
authors.lastname: Szakal
authors.affiliation: Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms, EN6 3LD, UK.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Lowther
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
authors.forename: Fanfan
authors.initials: F
authors.lastname: Hao
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA Programs in Biochemistry, Cell, and Molecular Biology, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021, USA.
authors.forename: Prabha
authors.initials: P
authors.lastname: Sarangi
authors.affiliation: IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Via Adamello 16, 20139, Italy.
authors.forename: Dana
authors.initials: D
authors.lastname: Branzei
authors.affiliation: Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms, EN6 3LD, UK.
authors.forename: Helle D
authors.initials: HD
authors.lastname: Ulrich
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA Programs in Biochemistry, Cell, and Molecular Biology, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021, USA zhaox1@mskcc.org.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Zhao
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D004249:DNA Damage; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D015151:Immunoblotting; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D044767:Ubiquitin-Protein Ligases; D000251:Adenosine Triphosphatases; C076225:RAD5 protein, S cerevisiae; D004265:DNA Helicases