Reconstitution of eukaryotic lagging strand DNA replication

Abstract

Eukaryotic DNA replication is a complex process requiring the proper functioning of a multitude of proteins to create error-free daughter DNA strands and maintain genome integrity. Even though synthesis and joining of Okazaki fragments on the lagging strand involves only half the DNA in the nucleus, the complexity associated with processing these fragments is about twice that needed for leading strand synthesis. Flap endonuclease 1 (FEN1) is the central component of the Okazaki fragment maturation pathway. FEN1 cleaves flaps that are displaced by DNA polymerase delta (pol delta), to create a nick that is effectively joined by DNA ligase I. The Pif1 helicase and Dna2 helicase/nuclease contribute to the maturation process by elongating the flap displaced by pol delta. Though the reason for generating long flaps is still a matter of debate, genetic studies have shown that Dna2 and Pif1 are both important components of DNA replication. Our current knowledge of the exact enzymatic steps that govern Okazaki fragment maturation has heavily derived from reconstitution reactions in vitro, which have augmented genetic information, to yield current mechanistic models. In this review, we describe both the design of specific DNA substrates that simulate intermediates of fragment maturation and protocols for reconstituting partial and complete lagging strand replication.

Fig 1. Pol dd d Primer Extension

(A) Model of Pol δ in complex with PCNA and RFC synthesizing in the 5′ – 3′ direction. (B) Model substrates containing biotin-conjugated streptavidin on the 5′ and 3′ ends of the template strand. Primer extension substrate model (i) and Okazaki fragment substrate or strand displacement synthesis substrate model (ii). (C) Assessment of primer extension. The asterisk indicates the position of the radiolabel.

Fig 2. FEN1 is structure specific nuclease

(A) FEN1 after it completes tracking to the base of a flap, and just before cleavage. (B) FEN1 flap cleavage is measured on a denaturing gel using an unblocked flap (left panel) and 5′ blocked flap (right panel) substrate. The asterisk indicates the position of the radiolabel. Fig 2B was originally published in Journal of Biological Chemistry by Murante R.S., et al., “Calf 5′ to 3′ Exo/Endonuclease Must Slide from a 5′ End of the Substrate to Perform Structure-specific Cleavage”, J Biol. Chem., (1995); 270:30377-30383 © the American Society for Biochemistry and Molecular Biology

Fig 3. Lig I seals DNA nicks

(A) Model of Lig I binding and sealing nicked DNA. (B) Denaturing gel assessing DNA Lig I ligation efficiency on a nicked DNA substrate. The asterisk indicates the position of the radiolabel.

Fig 4. RPA binds single stranded DNA and melts short double stranded DNA segments

(A) Model of RPA binding the single stranded DNA region of a flap (top arrow) or melting and binding the substrate (bottom arrow). (B) RPA binding is visualized using an EMSA. Lane 1 shows the labeled flap substrate as a control. Lanes 1 – 4 indicate RPA bound to intact substrate forming shifted complexes. Lanes 5 – 7 indicate RPA bound to melted substrate forming alternate shifted complexes. The asterisk indicates the position of the radiolabel.

Figure 5. Dna2 displaces RPA from ssDNA and cleaves at multiple sites on the ssDNA

(A) Model for Dna2 5′ – 3′ helicase unwinding. (B) Model for the Dna2 5′ – 3′ nuclease activity. Note that helicase activity may displace into the downstream primer, but the terminal nuclease product is always a 5-6 nt flap. (C) EMSA gel showing Dna2E675A (nuclease mutant) displacement of RPA from the 5′ flap. (D) Denaturing gel showing RPA stimulation of Dna2 cleavage. The asterisk indicates the position of the radiolabel. Figure 5C, D were originally published in Journal of Biological Chemistry by Stewart, J.A., et al., “Dynamic removal of replication protein A by Dna2 facilitates primer cleavage during Okazaki fragment processing in Saccharomyces cerevisiae”, J Biol. Chem., (2008); 283 (46):31356-65 © the American Society for Biochemistry and Molecular Biology

Fig 6. Pif1 can elongate the pol dd d displaced flap

(A) Model of the Pif1 5′ – 3′ helicase activity. (B) Native gel assessing Pif1 helicase activity on a 30 and 53 nt flap substrate. The asterisk indicates the position of the radiolabel.

Fig 7. Full Okazaki fragment processing reconstitution system

(A) Model of the two-nuclease pathway (see text for description of individual steps). (B) Denaturing gel assessing ligation efficiency of an Okazaki substrate in a reconstituted system. The asterisk indicates the position of the radiolabel. Figure 7B was originally published in Journal of Biological Chemistry by Pike, J.E., et al., “Pif1 helicase lengthens some Okazaki fragment flaps necessitating Dna2 nuclease/helicase action in the two-nuclease processing pathway”, J Biol. Chem., (2009); 284(37):25170-80 © the American Society for Biochemistry and Molecular Biology