(NAS Colloquium) Links Between Recombination and Replication: Vital Roles of Recombination

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Colloquium on Links Between Recombination and Replication: Vital Roles of Recombination

Colloquium

Bacteriophage T4 gene 41 helicase and gene 59 helicase-loading protein: A versatile couple with roles in replication and recombination

Charles E.Jones*, Timothy C.Mueser^†, Kathleen C.Dudas^‡, Kenneth N.Kreuzer^‡, and Nancy G.Nossal*^§

*Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892–0830; ^†Department of Chemistry, University of Toledo, 2801 West Bancroft Street, Toledo, OH 43606; and ^‡Department of Microbiology, Duke University Medical Center, Durham, NC 27710

Bacteriophage T4 uses two modes of replication initiation: origin-dependent replication early in infection and recombination-dependent replication at later times. The same relatively simple complex of T4 replication proteins is responsible for both modes of DNA synthesis. Thus the mechanism for loading the T4 41 helicase must be versatile enough to allow it to be loaded on R loops created by transcription at several origins, on D loops created by recombination, and on stalled replication forks. T4 59 helicase-loading protein is a small, basic, almost completely α-helical protein whose N-terminal domain has structural similarity to high mobility group family proteins. In this paper we review recent evidence that 59 protein recognizes specific structures rather than specific sequences. It binds and loads the helicase on replication forks and on three- and four-stranded (Holliday junction) recombination structures, without sequence specificity. We summarize our experiments showing that purified T4 enzymes catalyze complete unidirectional replication of a plasmid containing the T4 ori(uvsY) origin, with a preformed R loop at the position of the R loop identified at this origin in vivo. This replication depends on the 41 helicase and is strongly stimulated by 59 protein. Moreover, the helicase-loading protein helps to coordinate leading and lagging strand synthesis by blocking replication on the ori(uvsY) R loop plasmid until the helicase is loaded. The T4 enzymes also can replicate plasmids with R loops that do not have a T4 origin sequence, but only if the R loops are within an easily unwound DNA sequence.

The bacteriophage T4-infected cell is a replication factory designed for the rapid production of multiple copies of its genome. T4 replication does not need to be coordinated with cell division or a cell cycle. Instead, the T4 replication strategy is optimized for quickly producing and packaging its DNA. The phage uses two modes of replication initiation: origin-dependent replication early in infection and recombination-dependent replication at later times. Because the 168-kb linear phage genome is terminally redundant and circularly permuted, the end of one DNA molecule is homologous to the middle of another. Thus the products of early origin-dependent replication can invade each other to form D loops for recombination-initiated replication. The same complex of relatively few T4-encoded replication proteins is responsible for DNA synthesis in both modes of replication (reviewed in refs. 1–4).

The genes for each of the T4 replication proteins have been cloned, and the functions of the proteins have been characterized by in vitro reactions on model templates (Fig. 1) (reviewed in ref. 5). T4 DNA polymerase (gene 43), which catalyzes DNA synthesis on both leading and lagging strands, is attached to a clamp protein (gene 45) that is loaded by the complex of the gene 44 and 62 proteins. In the presence of the T4 gene 32 single-stranded DNA binding protein, T4 DNA polymerase, the clamp, and the clamp loader are sufficient for slow strand displacement synthesis of the leading strand. The 5′ to 3′ gene 41 helicase unwinds DNA ahead of the fork and increases the elongation rate more than 10-fold to 400 nt/sec, comparable to that in vivo. Although the helicase can load on nicked and forked DNA by itself, its loading is greatly accelerated by the 59 helicase-loading protein. Lagging strand fragments are initiated by pentamer RNA primers, whose synthesis requires both the helicase and gene 61 primase. These primers ultimately are removed by a T4 encoded 5′ to 3′ nuclease (T4 RNase H), and after gap repair, the adjacent fragments are joined by T4 DNA ligase. T4 type II topoisomerase (genes 39, 52, and 60) is required for replication in vitro when the parental duplex is a covalently closed circle.

In tightly controlled replication systems like Escherichia coli oriC or yeast ARS I, sequence-specific origin binding proteins help to load the helicase at the origin (6). In contrast, the seven known T4 replication origins, identified by a variety of methods, do not share DNA sequences (1, 3). The four best-characterized origins, oriA, oriE, ori(uvsY)(F), and ori(34)(G), each have a transcription promoter, whose transcript is thought to act as the primer for leading strand synthesis. With the exception of the repEB and repEA proteins at oriE (7), there is no evidence for T4-encoded proteins that are required for initiation at a specific origin. Consequently, the mechanism for loading the T4 41 helicase must be versatile enough to allow it to load on R loops created by transcription at several origins, on D loops created by recombination, and on stalled replication forks.

In this paper we review recent evidence demonstrating that the T4 gene 59 helicase-loading protein recognizes specific structures rather than specific sequences. It binds and loads the helicase on replication forks and on three- and four-stranded (Holliday junction) recombination structures, without sequence specificity (8, 9). We summarize our experiments showing that the purified T4 enzymes catalyze complete unidirectional replication of a plasmid containing T4 ori(uvsY), with a preformed R loop at the position of the R loop identified in vivo (10). This replication depends on the

This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA.

Abbreviations: HMG, high mobility group; PDB, Protein Data Bank; DUE, DNA-unwinding element.

^§	To whom reprint requests should be addressed at: Laboratory of Molecular and Cellular Biology, Building 8, Room 2A19, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892–0830. E-mail: ngn@helix.nih.gov.

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