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A Versatile Couple with Roles in Replication and Recombination

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A Versatile Couple with Roles in Replication and 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- protein (gene 45) that is loaded by the complex of the gene 44 dependent replication early in infection and recombination-depen- and 62 proteins. In the presence of the T4 gene 32 single- dent replication at later times. The same relatively simple complex stranded DNA binding protein, T4 DNA polymerase, the clamp, of T4 replication proteins is responsible for both modes of DNA and the clamp loader are sufficient for slow strand displacement synthesis. Thus the mechanism for loading the T4 41 helicase must synthesis of the leading strand. The 5Ј to 3Ј gene 41 helicase be versatile enough to allow it to be loaded on R loops created by unwinds DNA ahead of the fork and increases the elongation transcription at several origins, on D loops created by recombina- rate more than 10-fold to 400 nt͞sec, comparable to that in vivo. tion, and on stalled replication forks. T4 59 helicase-loading protein Although the helicase can load on nicked and forked DNA by is a small, basic, almost completely ␣-helical protein whose N- itself, its loading is greatly accelerated by the 59 helicase-loading terminal domain has structural similarity to high mobility group protein. Lagging strand fragments are initiated by pentamer family proteins. In this paper we review recent evidence that 59 RNA primers, whose synthesis requires both the helicase and protein recognizes specific structures rather than specific se- gene 61 primase. These primers ultimately are removed by a T4 quences. It binds and loads the helicase on replication forks and on encoded 5Ј to 3Ј nuclease (T4 RNase H), and after gap repair, three- and four-stranded (Holliday junction) recombination struc- the adjacent fragments are joined by T4 DNA ligase. T4 type II tures, without sequence specificity. We summarize our experi- topoisomerase (genes 39, 52, and 60) is required for replication ments showing that purified T4 enzymes catalyze complete uni- in vitro when the parental duplex is a covalently closed circle. directional replication of a plasmid containing the T4 ori(uvsY) In tightly controlled replication systems like Escherichia coli origin, with a preformed R loop at the position of the R loop oriC or yeast ARS I, sequence-specific origin binding proteins identified at this origin in vivo. This replication depends on the 41 help to load the helicase at the origin (6). In contrast, the seven helicase and is strongly stimulated by 59 protein. Moreover, the known T4 replication origins, identified by a variety of methods, helicase-loading protein helps to coordinate leading and lagging do not share DNA sequences (1, 3). The four best-characterized strand synthesis by blocking replication on the ori(uvsY) R loop origins, oriA, oriE, ori(uvsY)(F), and ori(34)(G), each have a plasmid until the helicase is loaded. The T4 enzymes also can transcription promoter, whose transcript is thought to act as the replicate plasmids with R loops that do not have a T4 origin primer for leading strand synthesis. With the exception of the sequence, but only if the R loops are within an easily unwound repEB and repEA proteins at oriE (7), there is no evidence for DNA sequence. 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 he bacteriophage T4-infected cell is a replication factory created by transcription at several origins, on D loops created by Tdesigned for the rapid production of multiple copies of its recombination, and on stalled replication forks. genome. T4 replication does not need to be coordinated with cell In this paper we review recent evidence demonstrating that the division or a cell cycle. Instead, the T4 replication strategy is T4 gene 59 helicase-loading protein recognizes specific structures optimized for quickly producing and packaging its DNA. The rather than specific sequences. It binds and loads the helicase on phage uses two modes of replication initiation: origin-dependent replication forks and on three- and four-stranded (Holliday junc- replication early in infection and recombination-dependent rep- tion) recombination structures, without sequence specificity (8, 9). lication at later times. Because the 168-kb linear phage genome We summarize our experiments showing that the purified T4 is terminally redundant and circularly permuted, the end of one enzymes catalyze complete unidirectional replication of a plasmid DNA molecule is homologous to the middle of another. Thus the containing T4 ori(uvsY), with a preformed R loop at the position of products of early origin-dependent replication can invade each the R loop identified in vivo (10). This replication depends on the 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 This paper results from the National Academy of Sciences colloquium, ‘‘Links Between replication (reviewed in refs. 1–4). Recombination and Replication: Vital Roles of Recombination,’’ held November 10–12, 2000, in Irvine, CA. The genes for each of the T4 replication proteins have been Abbreviations: HMG, high mobility group; PDB, Protein Data Bank; DUE, DNA-unwinding cloned, and the functions of the proteins have been characterized element. by in vitro reactions on model templates (Fig. 1) (reviewed in ref. §To whom reprint requests should be addressed at: Laboratory of Molecular and Cellular 5). T4 DNA polymerase (gene 43), which catalyzes DNA syn- Biology, Building 8, Room 2A19, National Institute of Diabetes and Digestive and Kidney thesis on both leading and lagging strands, is attached to a clamp Diseases, National Institutes of Health, Bethesda, MD 20892-0830. E-mail: [email protected]. 8312–8318 ͉ PNAS ͉ July 17, 2001 ͉ vol. 98 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.121009398 Downloaded by guest on September 26, 2021 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. T4 Gene 59 Helicase-Loading Protein T4 mutants in gene 59 are UV-sensitive and defective in repair, recombination, and the recombination-initiated replication pre- dominant in the late stage of infection (11, 12). The initial characterization of purified 59 protein showed that it was a small, monomeric, and basic protein that was capable of binding both single- and double-stranded DNA. 59 Protein also was shown to interact specifically with 41 helicase and gene 32 single-stranded DNA binding protein in the presence and absence of DNA (13–16). Barry and Alberts (16) showed that 59 protein increased the rate of loading of the 41 helicase on both single-stranded and nicked duplex templates. Its ability to load the helicase on single-stranded DNA covered with 32 protein was particularly striking because 32 protein strongly inhibits DNA unwinding by the helicase without 59 protein. Structure of 59 Protein. The crystal structure of the full-length (217 residues) 59 helicase-loading protein [Protein Data Bank (PDB) code 1C1K; ref. 8] revealed a novel, almost entirely ␣-helical protein, with no similarity to the structures of other single- stranded DNA binding proteins (Fig. 2). Its 13 ␣-helices are divided into N and C domains of similar size. The single short ␤-sheet connects N-terminal residues 2–4 with residues 197–199 near the C terminus. There is a narrow groove between the two domains on the ‘‘top’’ of the protein. The surface of the protein is notable for the high density of basic and hydrophobic residues, which may be important for its DNA and protein interactions. The sequence of 59 protein has only limited similarity to the sequences of functionally related proteins. However, the N domain of 59 protein has strong structural similarity to several members of Fig. 2. Ribbon diagrams of the crystal structure of the T4 gene 59 helicase- the high mobility group (HMG) family proteins, including rat loading protein showing its structural similarity with HMG proteins. (A) and HMG1A and the LEF-1 lymphoid enhancer-binding factor (8). The (B) are, respectively, ‘‘top’’ and ‘‘side’’ view rainbow-colored ribbon diagrams HMG domain has been found in structure-specific nonhistone of 59 protein (PDB 1C1K, ref. 8) from the blue N terminus to the red C terminus. chromatin-binding proteins and sequence-specific transcription fac- S1–2 are ␤-sheets and H1–13 are ␣-helices. The line between H6 and H7 tors. Proteins with the HMG domain have been shown to bind to indicates the junction between the N and C domains of the protein. (C) the minor groove of DNA, bending and partially unwinding the Superposition of the H1, H2, and H3 ␣-helices of T4 59 helicase-loading protein duplex. HMG1 and other family members bind and unstack cru- (blue) and rat HMG1 protein (green, PDB 1AAB, ref. 20) and with the DNA (red) from the model of lymphoid enhancer-binding factor LEF1-DNA, (PDB 2LEF, ciform DNA (reviewed in refs. 17–19). The structural alignment on ref. 21). Adapted from Mueser et al.
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