Dna2 nuclease-helicase structure, mechanism and regulation by Rpa.
The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5' end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5' but not 3' end, explaining how Rpa regulates cleavage polarity.
Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States.
Chun
C
Zhou
Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States.
Sergei
S
Pourmal
Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, United States.
Nikola P
NP
Pavletich
D000818:Animals; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D064251:Multifunctional Enzymes / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D051716:Replication Protein A / Q000378:metabolism*
D004277:DNA, Single-Stranded; D064251:Multifunctional Enzymes; D051716:Replication Protein A; C497168:Rpa1 protein, mouse; C000610111:Dna2 protein, mouse; D004706:Endodeoxyribonucleases; D004265:DNA Helicases
DNA end resection; DNA replication; Dna2; Rpa; biophysics; chromosomes; genes; helicase; none; nuclease; structural biology