The Rad54 protein seed DNA repair synthesis

KREJČÍ, Lumír. The Rad54 protein seed DNA repair synthesis. Amsterodam, 2009.

Základní údaje

• Originální název: The Rad54 protein seed DNA repair synthesis
• Název česky: Rad54 protein a jeho role při DNA opravné syntéze
• Autoři: KREJČÍ, Lumír.
• Vydání: Amsterodam, 2009.

Další údaje

• Originální jazyk: angličtina
• Typ výsledku: Audiovizuální tvorba
• Obor: 10600 1.6 Biological sciences
• Stát vydavatele: Nizozemské království
• Utajení: není předmětem státního či obchodního tajemství
• WWW: URL
• Organizační jednotka: Lékařská fakulta
• Klíčová slova anglicky: DNA repair; DNA damage; replication; genomic instability
• Příznaky: Mezinárodní význam

Anotace

Rad54 is a key enzyme involved in the genome maintenance pathway of homologous recombination (HR). This protein has been implicated in several steps of HR, but its exact role(s) at each step is still not fully understood. We have identified a novel interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction can be abrogated by mutation of two hydrophobic residues in the conserved PCNA-interaction motif (PIP-box). This rad54 mutant (rad54-AA) is specifically defective in PCNA binding, retaining its ability to interact with Rad51, to promote in vitro strand exchange activity, shows similar ATPase activity to the wild-type protein, and is able to be recruited to HR foci in vivo. Despite this proficiency, rad54-AA mutant cells display a phenotype similar to the null in recombination assays and sensitivity to DNA damaging agents. Furthermore, the rad54-AA mutant shows a phenotype consistent with mutants that have defects in late HR steps, such as increased Rad52 focus duration and synthetic lethality with srs2 deletion. Since this mutant specifically affects interactions with the PCNA replication clamp, we tested the role of this mutant during the repair synthesis step of HR. Indeed, the rad54 PIP-box mutant displays defects in primer extension at the MAT locus. This is further supported by observation that Rad54 stimulates extension activity by Poldelta in vitro, likely mediated by PCNA interactions, since the rad54-AA mutant curtails this stimulation. We suggest that Rad54-PCNA interactions are critical during late steps of HR, and for allowing efficient DNA repair synthesis to proceed.

Anotace česky

Rad54 is a key enzyme involved in the genome maintenance pathway of homologous recombination (HR). This protein has been implicated in several steps of HR, but its exact role(s) at each step is still not fully understood. We have identified a novel interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction can be abrogated by mutation of two hydrophobic residues in the conserved PCNA-interaction motif (PIP-box). This rad54 mutant (rad54-AA) is specifically defective in PCNA binding, retaining its ability to interact with Rad51, to promote in vitro strand exchange activity, shows similar ATPase activity to the wild-type protein, and is able to be recruited to HR foci in vivo. Despite this proficiency, rad54-AA mutant cells display a phenotype similar to the null in recombination assays and sensitivity to DNA damaging agents. Furthermore, the rad54-AA mutant shows a phenotype consistent with mutants that have defects in late HR steps, such as increased Rad52 focus duration and synthetic lethality with srs2 deletion. Since this mutant specifically affects interactions with the PCNA replication clamp, we tested the role of this mutant during the repair synthesis step of HR. Indeed, the rad54 PIP-box mutant displays defects in primer extension at the MAT locus. This is further supported by observation that Rad54 stimulates extension activity by Poldelta in vitro, likely mediated by PCNA interactions, since the rad54-AA mutant curtails this stimulation. We suggest that Rad54-PCNA interactions are critical during late steps of HR, and for allowing efficient DNA repair synthesis to proceed.

Návaznosti

• GA301/09/1917, projekt VaV: Název: Štěpení replikačních-rekombinačních DNA meziproduktů a jejich úloha při nestabilitě genomu

Investor: Grantová agentura ČR, Štěpení replikačních-rekombinačních DNA meziproduktů a jejich úloha při nestabilitě genomu

• GD203/09/H046, projekt VaV: Název: Biochemie na rozcestí mezi in silico a in vitro

Investor: Grantová agentura ČR, Biochemie na rozcestí mezi in silico a in vitro

• LC06030, projekt VaV: Název: Biomolekulární centrum

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Biomolekulární centrum

• MSM0021622413, záměr: Název: Proteiny v metabolismu a při interakci organismů s prostředím

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Proteiny v metabolismu a při interakci organismů s prostředím