Helicase Primase DNA Polymerases and Other Proteins Participate in DNA Replication

Detailed understanding of the eukaryotic proteins that participate in DNA replication has come largely from studies with small viral DNAs, particularly SV40 DNA, the circular genome of a small virus that infects monkeys. Figure 4-34 depicts the multiple proteins that coordinate copying of SV40 DNA at a replication fork. The assembled proteins at a replication fork further illustrate the concept of molecular machines introduced in Chapter 3. These multicomponent

▲ FIGURE 4-34 Model of an SV40 DNA replication fork and assembled proteins. (a) A hexamer of large T-antigen (D), a viral protein, functions as a helicase to unwind the parental DNA strands. Single-strand regions of the parental template unwound by large T-antigen are bound by multiple copies of the heterotrimeric protein RPA (2). The leading strand is synthesized by a complex of DNA polymerase 8 (Pol 8), PCNA, and Rfc (H). Primers for lagging-strand synthesis (red, RNA; light blue, DNA) are synthesized by a complex of DNA polymerase a (Pol a) and primase (4). The 3' end of each primer synthesized by Pol a-primase is then bound by a PCNA-Rfc-Pol 8 complex, which proceeds to extend the primer and synthesize most of each Okazaki fragment (0). See the text for details. (b) The three subunits of PCNA, shown in different colors, form a circular structure with a central hole through which double-stranded DNA passes. A diagram of DNA is shown in the center of a ribbon model of the PCNA trimer. (c) The large subunit of RPA contains two domains that bind single-stranded DNA. On the left, the two DNA-binding domains of RPA are shown perpendicular to the DNA backbone (white backbone with blue bases). Note that the single DNA strand is extended with the bases exposed, an optimal conformation for replication by a DNA polymerase. On the right, the view is down the length of the single DNA strand, revealing how RPA ß strands wrap around the DNA. [Part (a) adapted from S. J. Flint et al., 2000, Virology: Molecular Biology, Pathogenesis, and Control, ASM Press; part (b) after J. M. Gulbis et al., 1996, Cell 87:297; and part (c) after A. Bochkarev et al., 1997, Nature 385:176.]

complexes permit the cell to carry out an ordered sequence of events that accomplish essential cell functions.

In the molecular machine that replicates SV40 DNA, a hexamer of a viral protein called large T-antigen unwinds the parental strands at a replication fork. All other proteins involved in SV40 DNA replication are provided by the host cell. Primers for leading and lagging daughter-strand DNA are synthesized by a complex of primase, which synthesizes a short RNA primer, and DNA polymerase a (Pol a), which extends the RNA primer with deoxynucleotides, forming a mixed RNA-DNA primer.

The primer is extended into daughter-strand DNA by DNA polymerase 8 (Pol 8), which is less likely to make errors during copying of the template strand than is Pol a. Pol 8 forms a complex with Rfc (replication factor C) and PCNA (proliferating cell nuclear antigen), which displaces the primase-Pol a complex following primer synthesis. As illustrated in Figure 4-34b, PCNA is a homotrimeric protein that has a central hole through which the daughter duplex DNA passes, thereby preventing the PCNA-Rfc-Pol 8 complex from dissociating from the template.

After parental DNA is separated into single-stranded templates at the replication fork, it is bound by multiple copies of RPA (replication protein A), a heterotrimeric protein (Figure 4-34c). Binding of RPA maintains the template in a uniform conformation optimal for copying by DNA polymerases. Bound RPA proteins are dislodged from the parental strands by Pol a and Pol 8 as they synthesize the complementary strands base-paired with the parental strands.

Several eukaryotic proteins that function in DNA replication are not depicted in Figure 4-34. A topoisomerase associates with the parental DNA ahead of the helicase to remove torsional stress introduced by the unwinding of the parental strands. Ribonuclease H and FEN I remove the ribonucleotides at the 5' ends of Okazaki fragments; these are replaced by deoxynucleotides added by DNA polymerase 8 as it extends the upstream Okazaki fragment. Successive Okazaki fragments are coupled by DNA ligase through standard 5'n3' phosphoester bonds.

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