Xenopus DNA2 is a helicase/nuclease that is found in complexes with replication proteins And-1/Ctf4 and Mcm10 and DSB response proteins Nbs1 and ATM.
We have used the Xenopus laevis egg extract system to study the roles of vertebrate Dna2 in DNA replication and double-strand-break (DSB) repair.  We first establish that Xenopus Dna2 is a helicase, as well as a nuclease.  We further show that Dna2 is a nuclear protein that is actively recruited to DNA only after replication origin licensing.  Dna2 co-localizes in foci with RPA and is found in a complex with replication fork components And-1 and Mcm10.  Dna2 interacts with the DSB repair and checkpoint proteins Nbs1 and ATM.  We also determine the order of arrival of ATM, MRN, Dna2, TopBP1, and RPA to duplex DNA ends and show that it is the same both in S phase and M phase extracts.  Interestingly, Dna2 can bind to DNA ends independently of MRN, but efficient nucleolytic resection, as measured by RPA recruitment, requires both MRN and Dna2.  The nuclease activity of Mre11 is required, since its inhibition delays both full Dna2 recruitment and resection. Dna2 depletion inhibits but does not block resection, and Chk1 and Chk2 induction occurs in the absence of Dna2.
Division of Biology, California Institute of Technology, Pasadena, CA, USA.
Karen E
KE
Wawrousek
Barbara K
BK
Fortini
Piotr
P
Polaczek
Lu
L
Chen
Qingquan
Q
Liu
William G
WG
Dunphy
Judith L
JL
Campbell
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D002457:Cell Extracts; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D006801:Humans; D064110:Minichromosome Maintenance Proteins; D008938:Mitosis; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016196:S Phase; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D029867:Xenopus Proteins / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002457:Cell Extracts; D002843:Chromatin; D004268:DNA-Binding Proteins; C527642:Mcm10 protein, Xenopus; D046912:Multiprotein Complexes; C511600:NBN protein, Xenopus; D025521:Tumor Suppressor Proteins; C106931:WDHD1 protein, Xenopus; D029867:Xenopus Proteins; D004247:DNA; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D004720:Endonucleases; D004265:DNA Helicases; C533186:DNA2 protein, Xenopus; D064110:Minichromosome Maintenance Proteins