6774–6786 Nucleic Acids Research, 2012, Vol. 40, No. 14 doi:10.1093/nar/gks388 Published online 7 May 2012 Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1 Jason W. Gloor1, Lata Balakrishnan 1 , Judith L. Campbell2 and Robert A. Bambara1,* 1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 and 2Braun Laboratories, California Institute of Technology, Pasadena, CA 91125, USA Received March 12, 2012; Revised April 5, 2012; Accepted April 13, 2012 ABSTRACT In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical tech- niques, binding and cleavage assays presented here indicate that when the flap is 30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonucle- ase (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation. INTRODUCTION In both prokaryotic and eukaryotic DNA replication, one strand of DNA, called lagging, is initially made in discon- tinuous segments termed Okazaki fragments. In humans, each fragment is initiated by an RNA primer 8–10 nt in length and extended 150–200 nt with DNA (1). The fragments need to be joined to make a continuous strand. This involves strand displacement synthesis from one fragment to raise the RNA primer region of the adjacent fragment into a ﬂap. Genetic experiments and reconstitutions in vitro suggest that ﬂap creation and removal occurs via two pathways. One employs the ﬂap endonuclease (FEN1), which removes the ﬂap containing the initiator RNA leaving a nick for ligation. If a ﬂap escapes FEN1 to grow long enough to acquire the eukary- otic DNA-binding protein (RPA), nucleolytic cleavage will be blocked. If so, the ﬂap is processed by the second pathway in which the nuclease/helicase Dna2 displaces the RPA. Dna2 then successively cleaves the ﬂap until it is too short to bind RPA and becomes a substrate for FEN1 (2,3). Biochemical analyses have demonstrated that both FEN1 (4–13) and Dna2 (14) employ a threading mechan- ism for binding and cleavage of the ﬂap substrate. Recent results show that both nucleases recognize and bind to the base of the ﬂap and then thread the 50 end of the ﬂap through the protein (15–17). Dna2 cleaves periodically during the threading (18,19), and FEN1 cleaves speciﬁc- ally at the base (4,20,21). Previous analyses with puriﬁed proteins from Saccharo- myces cerevisiae clariﬁed how these proteins act in succes- sion to process Okazaki fragments (8,22–25). On a long ﬂap substrate, S. cerevisiae Dna2 (scDna2) was shown to displace bound scRPA in a reaction that did not require cleavage (26). scFEN1 was shown to displace the scDna2, so that it could have access to its cleavage site at the ﬂap base (17). In addition to the role it plays in Okazaki fragment pro- cessing, Dna2 functions in conjunction with 30–50 helicases and RPA to resect the 50 end of DNA during double-strand break (DSB) repair and telomere maintenance (27). Human and yeast systems employ redundant pathways during these processes, one of which utilizes Dna2 (28–30). Once recruited to the DSB, a 30–50 helicase separ- ates the two strands of the DNA creating a pseudo-Y struc- ture consisting of a 30 single-stranded (ss) DNA tail, a 50 ssDNA tail and a double-stranded region. These single- stranded tails can then be coated by RPA. Similar to its role in the long ﬂap Okazaki fragment pathway, Dna2 cleaves the 50-ssDNA. Surprisingly, even though Dna2 possesses a 30 nuclease activity, RPA prevents cleavage of the 30-ssDNA (30,31). Dna2 interacts with the RPA1 (70 kDa) subunit of RPA (22). The N terminus of RPA1 *To whom correspondence should be addressed. Tel: +1 585 275 3269; Fax: +1 585 275 6007; Email: robert_bambara@urmc.rochester.edu The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.