Identification and mapping of protein-protein interactions between gp32 and gp59 by cross-linking

The bacteriophage T4 59 protein (gp59) plays a vital role in recombination and replication by promoting the assembly of the gene 41 helicase (gp41) onto DNA, thus enabling replication as well as strand exchange in recombination. Loading of the helicase onto gp32 (the T4 single strand binding protein)-coated single-stranded DNA requires gp59 to remove gp32 and replace it with gp41. Cross-linking studies between gp32 and gp59 reveal an interaction between Cys-166 of gp32 and Cys-42 of gp59. Since Cys-166 lies in the DNA binding core domain of gp32, this interaction may affect the association of gp32 with DNA. In the presence of gp32 or DNA, gp59 is capable of forming a multimer consisting of at least five gp59 subunits. Kinetics studies suggest that gp59 and gp41 exist in a one-to-one ratio, predicting that gp59 is capable of forming a hexamer (Raney, K. D., Carver, T. E., and Benkovic, S. J. (1996) J. Biol. Chem. 271, 14074-14081). The C-terminal A-domain of gp32 is needed for gp59 oligomer formation. Cross-linking has established that gp59 can interact with gp32-A (a truncated form of gp32 lacking the A-domain) but cannot form higher species. The results support a model in which gp59 binds to gp32 on a replication fork, destabilizing the gp32-single-stranded DNA interaction concomitant with the oligomerization of gp59 that results in a switching of gp41 for gp32 at the replication fork.     

Mutational analysis of the T4 gp59 helicase loader reveals its sites for interaction with helicase, single-stranded binding protein, and DNA

Efficient DNA replication involves coordinated interactions among DNA polymerase, multiple factors, and the DNA. From bacteriophage T4 to eukaryotes, these factors include a helicase to unwind the DNA ahead of the replication fork, a single-stranded binding protein (SSB) to bind to the ssDNA on the lagging strand, and a helicase loader that associates with the fork, helicase, and SSB. The previously reported structure of the helicase loader in the T4 system, gene product (gp)59, has revealed an N-terminal domain, which shares structural homology with the high mobility group (HMG) proteins from eukaryotic organisms. Modeling of this structure with fork DNA has suggested that the HMG-like domain could bind to the duplex DNA ahead of the fork, whereas the C-terminal portion of gp59 would provide the docking sites for helicase (T4 gp41), SSB (T4 gp32), and the ssDNA fork arms. To test this model, we have used random and targeted mutagenesis to generate mutations throughout gp59. We have assayed the ability of the mutant proteins to bind to fork, primed fork, and ssDNAs, to interact with SSB, to stimulate helicase activity, and to function in leading and lagging strand DNA synthesis. Our results provide strong biochemical support for the role of the N-terminal gp59 HMG motif in fork binding and the interaction of the C-terminal portion of gp59 with helicase and SSB. Our results also suggest that processive replication may involve the switching of gp59 between its interactions with helicase and SSB.     

Helicase assembly protein Gp59 of bacteriophage T4: fluorescence anisotropy and sedimentation studies of complexes formed with derivatives of Gp32, the phage ssDNA binding protein.

The gene 59 protein (gp59) of bacteriophage T4 performs a vital function in phage DNA replication by directing the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA at nascent replication forks. The helicase assembly activity of gp59 is required for optimum efficiency of helicase acquisition by the replication fork during strand displacement DNA synthesis and is essential for helicase and primosome assembly during T4 recombination-dependent DNA replication transactions. Of central importance is the ability of gp59 to load the gp41 helicase onto ssDNA previously coated with cooperatively bound molecules of gp32, the T4 ssDNA binding protein. Gp59 heteroassociations with ssDNA, gp32, and gp41 all appear to be essential for this loading reaction. Previous studies demonstrated that a tripartite complex containing gp59 and gp32 simultaneously cooccupying ssDNA is an essential intermediate in gp59-dependent helicase loading; however, the biochemical and structural parameters of gp59-gp32 complexes with or without ssDNA are currently unknown. To better understand gp59-gp32 interactions, we performed fluorescence anisotropy and analytical ultracentrifugation experiments employing native or rhodamine-labeled gp59 species in combination with altered forms of gp32, allowing us to determine their binding parameters, shape parameters, and other hydrodynamic properties. Two truncated forms of gp32 were used: gp32-B, which lacks the N-terminal B-domain required for cooperative binding to ssDNA and for stable self-association, and A-domain fragment, which is the C-terminal peptide of gp32 lacking ssDNA binding ability. Results indicate that gp59 binds with high affinity to either gp32 derivative to form a 1:1 heterodimer. In both cases, heterodimer formation is accompanied by a conformational change in gp59 which correlates with decreased gp59-DNA binding affinity. Hydrodynamic modeling suggests an asymmetric prolate ellipsoid shape for gp59, consistent with its X-ray crystallographic structure, and this asymmetry appears to increase upon binding of gp32 derivatives. Implications of our findings for the structure and function of gp59 and gp59-gp32 complexes in T4 replication are discussed.     

Simultaneous interactions of bacteriophage T4 DNA replication proteins gp59 and gp32 with single-stranded (ss) DNA. Co-modulation of ssDNA binding activities in a DNA helicase assembly intermediate.

The T4 gp59 protein is the major accessory protein of the phage's replicative DNA helicase, gp41. gp59 helps load gp41 at DNA replication forks by promoting its assembly onto single-stranded (ss) DNA covered with cooperatively bound molecules of gp32, the T4 single-strand DNA binding protein (ssb). A gp59-gp32-ssDNA ternary complex is an obligatory intermediate in this helicase loading mechanism. Here, we characterize the properties of gp59-gp32-ssDNA complexes and reveal some of the biochemical interactions that occur within them. Our results indicate the following: (i) gp59 is able to co-occupy ssDNA pre-saturated with either gp32 or gp32-A (a truncated gp32 species lacking interactions with gp59); (ii) gp59 destabilizes both gp32-ssDNA and (gp32-A)-ssDNA interactions; (iii) interactions of gp59 with the A-domain of gp32 alter the ssDNA-binding properties of gp59; and (iv) gp59 organizes gp32-ssDNA versus (gp32-A)-ssDNA into morphologically distinct complexes. Our results support a model in which gp59-gp32 interactions are non-essential for the co-occupancy of both proteins on ssDNA but are essential for the formation of structures competent for helicase assembly. The data argue that specific "cross-talk" between gp59 and gp32, involving conformational changes in both, is a key feature of the gp41 helicase assembly pathway.     

Interaction between the T4 helicase loading protein (gp59) and the DNA polymerase (gp43): unlocking of the gp59-gp43-DNA complex to initiate assembly of a fully functional replisome

Single-molecule fluorescence resonance energy transfer and functional assays have been used to study the initiation and regulation of the bacteriophage T4 DNA replication system. Previous work has demonstrated that a complex of the helicase loading protein (gp59) and the DNA polymerase (gp43) on forked DNA totally inhibits the polymerase and exonuclease activities of gp43 by a molecular locking mechanism (Xi, J., Zhuang, Z., Zhang, Z., Selzer, T., Spiering, M. M., Hammes, G. G., and Benkovic, S. J. (2005) Biochemistry 44, 2305-2318). We now show that this complex is "unlocked" by the addition of the helicase (gp41) with restoration of the DNA polymerase activity. Gp59 retains its ability to load the helicase while forming a gp59-gp43 complex at a DNA fork in the presence of the single-stranded DNA binding protein (gp32). Upon the addition of gp41 and MgATP, gp59 dissociates from the complex, and the DNA-bound gp41 is capable of recruiting the primase (gp61) to form a functional primosome and, subsequently, a fully active replisome. Functional assays of leading- and lagging-strand synthesis on an active replication fork show that the absence of gp59 has no effect on the coupling of leading- and lagging-strand synthesis or on the size of the Okazaki DNA fragments. We conclude that gp59 acts in a manner similar to the clamp loader to ensure proper assembly of the replisome and does not remain as a replisome component during active replication.     

Assembly and dynamics of Gp59-Gp32-single-stranded DNA (ssDNA), a DNA helicase loading complex required for recombination-dependent replication in bacteriophage T4

The Gp59 protein of bacteriophage T4 plays critical roles in recombination-dependent DNA replication and repair by correctly loading the replicative helicase, Gp41, onto recombination intermediates. Previous work demonstrated that Gp59 is required to load helicase onto single-stranded DNA that is saturated with Gp32, the T4 single-stranded DNA (ssDNA)-binding protein. Gp59 and Gp32 bind simultaneously to ssDNA, forming a Gp59-Gp32-ssDNA complex that is a key intermediate in helicase loading. Here we characterize the assembly and dynamics of this helicase loading complex (HLC) through changes in the fluorescent states of Gp32F, a fluorescein-Gp32 conjugate. Results show that HLC formation requires a minimum Gp32-ssDNA cluster size and that Gp59 co-localizes with Gp32-ssDNA clusters in the presence of excess free ssDNA. These and other results indicate that Gp59 targets helicase assembly onto Gp32-ssDNA clusters that form on the displaced strand of D-loops, which suggests a mechanism for the rapid initiation of recombination-dependent DNA replication. Helicase loading at the HLC requires ATP binding (not hydrolysis) by Gp41 and results in local remodeling of Gp32 within the HLC. Subsequent ATPase-driven translocation of Gp41 progressively disrupts Gp32-ssDNA interactions. Evidence suggests that Gp59 from the HLC is recycled to promote multiple rounds of helicase assembly on Gp32-ssDNA, a capability that could be important for the restart of stalled replication forks.     

Mechanistic studies of the T4 DNA (gp41) replication helicase: functional interactions of the C-terminal Tails of the helicase subunits with the T4 (gp59) helicase loader protein

We compare the activities of the wild-type (gp41WT) and mutant (gp41delta C20) forms of the bacteriophage T4 replication helicase. In the gp41delta C20 mutant the helicase subunits have been genetically truncated to remove the 20 residue C-terminal tail peptide domains present in the wild-type enzyme. Here, we examine the interactions of these helicase forms with the T4 gp59 helicase loader and the gp32 single-stranded DNA binding proteins, both of which are physically and functionally coupled with the helicase in the T4 DNA replication complex. We show that the wild-type and mutant forms of the helicase do not differ in their ability to assemble into dimers and hexamers, nor in their interactions with gp61 (the T4 primase). However they do differ in their gp59-stimulated unwinding activities and in their abilities to translocate along a ssDNA strand that has been coated with gp32. We demonstrate that functional coupling between gp59 and gp41 involves direct interactions between the C-terminal tail peptides of the helicase subunits and the loading protein, and measure the energetics and kinetics of these interactions. This work helps to define a gp41-gp59 assembly pathway that involves an initial interaction between the C-terminal tails of the helicases and the gp59 loader proteins, followed by a conformational change of the helicase subunits that exposes new interaction surfaces, which can then be trapped by the gp59 protein. Our results suggest that the gp41-gp59 complex is then poised to bind ssDNA portions of the replication fork. We suggest that one of the important functions of gp59 may be to aid in the exposure of the ssDNA binding sites of the helicase subunits, which are otherwise masked and regulated by interactions with the helicase carboxy-terminal tail peptides.     

Assembly of the bacteriophage T4 helicase: architecture and stoichiometry of the gp41-gp59 complex

The bacteriophage T4 59 protein (gp59) plays an essential role in recombination and replication by mediating the assembly of the gene 41 helicase (gp41) onto DNA. gp59 is required to displace the gp32 single-stranded binding protein on the lagging strand to expose a site for helicase binding. To gain a better understanding of the mechanism of helicase assembly, the architecture and stoichiometry of the gp41-gp59 complex were investigated. Both the N and C termini of gp41 were found to lie close to or in the gp41-gp41 subunit interface and interact with gp59. The site of interaction of gp41 on gp59 is proximal to Cys-215 of gp59. Binding of gp41 to gp59 stimulates a conformational change in the protein resulting in hexamer formation of gp59, and gp59 likewise stimulates oligomer formation of gp41. The gp59 subunits in this complex are arranged in a head to head orientation, such that Cys-42 of one subunit is in close proximity to Cys-42 on an adjacent subunit, and Cys-215 on one subunit is close to Cys-215 on a neighboring subunit. As the helicase is loaded onto DNA, a conformational change in the gp41-gp59 complex occurs, which may serve to displace gp32 from the lagging strand and load the hexameric helicase in its place.     

Dual functions of single-stranded DNA-binding protein in helicase loading at the bacteriophage T4 DNA replication fork

Semi-conservative DNA synthesis reactions catalyzed by the bacteriophage T4 DNA polymerase holoenzyme are initiated by a strand displacement mechanism requiring gp32, the T4 single-stranded DNA (ssDNA)-binding protein, to sequester the displaced strand. After initiation, DNA helicase acquisition by the nascent replication fork leads to a dramatic increase in the rate and processivity of leading strand DNA synthesis. In vitro studies have established that either of two T4-encoded DNA helicases, gp41 or dda, is capable of stimulating strand displacement synthesis. The acquisition of either helicase by the nascent replication fork is modulated by other protein components of the fork including gp32 and, in the case of the gp41 helicase, its mediator/loading protein gp59. Here, we examine the relationships between gp32 and the gp41/gp59 and dda helicase systems, respectively, during T4 replication using altered forms of gp32 defective in either protein-protein or protein-ssDNA interactions. We show that optimal stimulation of DNA synthesis by gp41/gp59 helicase requires gp32-gp59 interactions and is strongly dependent on the stability of ssDNA binding by gp32. Fluorescence assays demonstrate that gp59 binds stoichiometrically to forked DNA molecules; however, gp59-forked DNA complexes are destabilized via protein-protein interactions with the C-terminal "A-domain" fragment of gp32. These and previously published results suggest a model in which a mobile gp59-gp32 cluster bound to lagging strand ssDNA is the target for gp41 helicase assembly. In contrast, stimulation of DNA synthesis by dda helicase requires direct gp32-dda protein-protein interactions and is relatively unaffected by mutations in gp32 that destabilize its ssDNA binding activity. The latter data support a model in which protein-protein interactions with gp32 maintain dda in a proper active state for translocation at the replication fork. The relationship between dda and gp32 proteins in T4 replication appears similar to the relationship observed between the UL9 helicase and ICP8 ssDNA-binding protein in herpesvirus replication.     

Bacteriophage T4 helicase loader protein gp59 functions as gatekeeper in origin-dependent replication in vivo

Bacteriophage T4 initiates origin-dependent replication via an R-loop mechanism in vivo. During in vitro reactions, the phage-encoded gp59 stimulates loading of the replicative helicase, gp41, onto branched intermediates, including origin R-loops. However, although gp59 is essential for recombination-dependent replication from D-loops, it does not appear to be required for origin-dependent replication in vivo. In this study, we have analyzed the origin-replicative intermediates formed during infections that are deficient in gp59 and other phage replication proteins. During infections lacking gp59, the initial replication forks from two different T4 origins actively replicated both leading- and lagging-strands. However, the retrograde replication forks from both origins were abnormal in the gp59-deficient infections. The lagging-strand from the initial fork was elongated as a new leading-strand in the retrograde direction without lagging-strand synthesis, whereas in the wild-type, leading- and lagging-strand synthesis appeared to be coupled. These results imply that gp59 inhibits the polymerase holoenzyme in vivo until the helicase-primase (gp41-gp61) complex is loaded, and we thereby refer to gp59 as a gatekeeper. We also found that all origin-replicative intermediates were absent in infections deficient in the helicase gp41 or the single-strand-binding protein gp32, regardless of whether gp59 was present or absent. These results argue that replication from the origin in vivo is dependent on both the helicase and single-strand-binding protein and demonstrate that the strong replication defect of gene 41 and 32 single mutants is not caused by gp59 inhibition of the polymerase.     

A zinc ribbon protein in DNA replication: primer synthesis and macromolecular interactions by the bacteriophage T4 primase.

The gene product 61 primase protein from bacteriophage T4 was expressed as an intein fusion and purified to homogeneity. The primase binds one zinc ion, which is coordinated by four cysteine residues to form a zinc ribbon motif. Factors that influence the rate of priming were investigated, and a physiologically relevant priming rate of approximately 1 primer per second per primosome was achieved. Primase binding to the single-stranded binding protein (1 primase:4 gp32 monomers; K(d) approximately 860 nM) and to the helicase protein in the presence of DNA and ATP-gamma-S (1 primase:1 helicase monomer; K(d) approximately 100 nM) was investigated by isothermal titration calorimetry (ITC). Because the helicase is hexameric, the inferred stoichiometry of primase binding as part of the primosome is helicase hexamer:primase in a ratio of 1:6, suggesting that the active primase, like the helicase, might have a ring-like structure. The primase is a monomer in solution but binds to single-stranded DNA (ssDNA) primarily as a trimer (K(d) approximately 50-100 nM) as demonstrated by ITC and chemical cross-linking. Magnesium is required for primase-ssDNA binding. The minimum length of ssDNA required for stable binding is 22-24 bases, although cross-linking reveals transient interactions on oligonucleotides as short as 8 bases. The association is endothermic at physiologically relevant temperatures, which suggests an overall gain in entropy upon binding. Some possible sources of this gain in entropy are discussed.     

Heterohexamer of 56- and 63-kDa Gene 4 Helicase-Primase of Bacteriophage T7 in DNA Replication

Bacteriophage T7 expresses two forms of gene 4 protein (gp4). The 63-kDa full-length gp4 contains both the helicase and primase domains. T7 phage also express a 56-kDa truncated gp4 lacking the zinc binding domain of the primase; the protein has helicase activity but no DNA-dependent primase activity. Although T7 phage grow better when both forms are present, the role of the 56-kDa gp4 is unknown. The two molecular weight forms oligomerize by virtue of the helicase domain to form heterohexamers. The 56-kDa gp4 and any mixture of 56- and 63-kDa gp4 show higher helicase activity in DNA unwinding and strand-displacement DNA synthesis than that observed for the 63-kDa gp4. However, single-molecule measurements show that heterohexamers have helicase activity similar to the 63-kDa gp4 hexamers. In oligomerization assays the 56-kDa gp4 and any mixture of the 56- and 63-kDa gp4 oligomerize to form more hexamers than does the 63-kDa gp4. The zinc binding domain of the 63-kDa gp4 interferes with hexamer formation, an inhibition that is relieved by the insertion of the 56-kDa species. Compared with the 63-kDa gp4, heterohexamers synthesize a reduced amount of oligoribonucleotides, mediated predominately by the 63-kDa subunits via a cis mode. During coordinated DNA synthesis 7% of the tetraribonucleotides synthesized are used as primers by both heterohexamers and hexamers of the 63-kDa gp4. Overall, an equimolar mixture of the two forms of gp4 shows the highest rate of DNA synthesis during coordinated DNA synthesis.     

The N-terminal 31 amino acids of human immunodeficiency virus type 1 envelope protein gp120 contain a potential gp41 contact site.

We have compared the expression of full-length gp160 envelope protein from human immunodeficiency virus type 1 with that of a deletion mutant lacking the N-terminal 31 amino acids of the mature protein (gp160 delta 32). The gp160 and gp160 delta 32 proteins are processed to yield gp41 and gp120 or gp120 delta 32, respectively. In contrast to full-length gp120, gp120 delta 32 failed to associate with gp41 at the cell surface, despite conformational integrity as judged by soluble CD4 binding. Thus, the N-terminal 31 amino acids of gp120, which contain hyperconserved sequences, are likely involved in forming a contact site for gp41.     

The structure of the unliganded extracellular domain of the interleukin-6 signal transducer gp130 in solution

Interleukin-6 (IL-6) plays an important role in immune responses and signals via two different pathways. When IL-6 binds to its non-signalling membrane-bound receptor (IL-6R), a non-covalent dimer of the ubiquitous interleukin-6 signal transducer gp130 is recruited to initiate intracellular signalling cascades. This so-called classical signalling pathway is restricted to cells expressing the membrane-bound IL-6R, such as hepatocytes and certain leukocytes. In addition, an alternative trans-signalling pathway uses soluble forms of IL-6R (sIL-6R) in complex with IL-6 to activate cells expressing gp130, but not membrane-bound IL-6R. In both cases, a tetrameric or hexameric signalling complex consisting of two gp130 molecules and one or two molecules each of IL-6 and (s)IL-6R is formed. The structure of the hexameric complex of the ligand-binding domains of gp130 (D1-D3) with IL-6 and sIL-6R has been solved by X-ray crystallography as well as the full-length extracellular part of gp130 (D1-D6) as a monomer. Since gp130 exists as a preformed dimer on the cell surface, we used a sgp130Fc fusion protein - consisting of two extracellular gp130 regions (D1-D6) dimerised by an IgG1-Fc part - to study the structure of unliganded gp130 extracellular domains in solution by small-angle X-ray scattering (SAXS). The SAXS data indicated that sgp130Fc forms a rigid molecule in solution. The low resolution structural model reveals an elongated assembly with an Fc base and two gp130 arms, whereby the orientation of the ligand-binding domains D1-D3 with respect to the membrane-proximal domains D4-D6 differs from that in the crystallographic monomer. Functional implications of these findings are discussed.     

Control of Helicase Loading in the Coupled DNA Replication and Recombination Systems of Bacteriophage T4

The Gp59 protein of bacteriophage T4 promotes DNA replication by loading the replicative helicase, Gp41, onto replication forks and recombination intermediates. Gp59 also blocks DNA synthesis by Gp43 polymerase until Gp41 is loaded, ensuring that synthesis is tightly coupled to unwinding. The distinct polymerase blocking and helicase loading activities of Gp59 likely involve different binding interactions with DNA and protein partners. Here, we investigate how interactions of Gp59 with DNA and Gp32, the T4 single-stranded DNA (ssDNA)-binding protein, are related to these activities. A previously characterized mutant, Gp59-I87A, exhibits markedly reduced affinity for ssDNA and pseudo-fork DNA substrates. We demonstrate that on Gp32-covered ssDNA, the DNA binding defect of Gp59-I87A is not detrimental to helicase loading and translocation. In contrast, on pseudo-fork DNA the I87A mutation is detrimental to helicase loading and unwinding in the presence or absence of Gp32. Other results indicate that Gp32 binding to lagging strand ssDNA relieves the blockage of Gp43 polymerase activity by Gp59, whereas the inhibition of Gp43 exonuclease activity is maintained. Our findings suggest that Gp59-Gp32 and Gp59-DNA interactions perform separate but complementary roles in T4 DNA metabolism; Gp59-Gp32 interactions are needed to load Gp41 onto D-loops, and other nucleoprotein structures containing clusters of Gp32. Gp59-DNA interactions are needed to load Gp41 onto nascent or collapsed replication forks lacking clusters of Gp32 and to coordinate bidirectional replication from T4 origins. The dual functionalities of Gp59 allow it to promote the initiation or re-start of DNA replication from a wide variety of recombination and replication intermediates.     

Site-directed mutations of T4 helicase loading protein (gp59) reveal multiple modes of DNA polymerase inhibition and the mechanism of unlocking by gp41 helicase

The T4 helicase loading protein (gp59) interacts with a multitude of DNA replication proteins. In an effort to determine the functional consequences of these protein-protein interactions, point mutations were introduced into the gp59 protein. Mutations were chosen based on the available crystal structure and focused on hydrophobic residues with a high degree of solvent accessibility. Characterization of the mutant proteins revealed a single mutation, Y122A, which is defective in polymerase binding and has weakened affinity for the helicase. The interaction between single-stranded DNA-binding protein and Y122A is unaffected, as is the affinity of Y122A for DNA substrates. When standard concentrations of helicase are employed, Y122A is unable to productively load the helicase onto forked DNA substrates. As a result of the loss of polymerase binding, Y122A cannot inhibit the polymerase during nucleotide idling or prevent it from removing the primer strand of a D-loop. However, Y122A is capable of inhibiting strand displacement synthesis by polymerase. The retention of strand displacement inhibition by Y122A, even in the absence of a gp59-polymerase interaction, indicates that there are two modes of polymerase inhibition by gp59. Inhibition of the polymerase activity only requires gp59 to bind to the replication fork, whereas inhibition of the exonuclease activity requires an interaction between the polymerase and gp59. The inability of Y122A to interact with both the polymerase and the helicase suggests a mechanism for polymerase unlocking by the helicase based on a direct competition between the helicase and polymerase for an overlapping binding site on gp59.     

Homologous recombination-dependent rescue of deficiency in the structural maintenance of chromosomes (Smc) 5/6 complex

The essential and evolutionarily conserved Smc5-Smc6 complex (Smc5/6) is critical for the maintenance of genome stability. Partial loss of Smc5/6 function yields several defects in DNA repair, which are rescued by inactivation of the homologous recombination (HR) machinery. Thus HR is thought to be toxic to cells with defective Smc5/6. Recent work has highlighted a role for Smc5/6 and the Sgs1 DNA helicase in preventing the accumulation of unresolved HR intermediates. Here we investigate how deletion of MPH1, encoding the orthologue of the human FANCM DNA helicase, rescues the DNA damage sensitivity of smc5/6 but not sgs1Δ mutants. We find that MPH1 deletion diminishes accumulation of HR intermediates within both smc5/6 and sgs1Δ cells, suggesting that MPH1 deletion is sufficient to decrease the use of template switch recombination (TSR) to bypass DNA lesions. We further explain how avoidance of TSR is nonetheless insufficient to rescue defects in sgs1Δ mutants, by demonstrating a requirement for Sgs1, along with the post-replicative repair (PRR) and HR machinery, in a pathway that operates in mph1Δ mutants. In addition, we map the region of Mph1 that binds Smc5, and describe a novel allele of MPH1 encoding a protein unable to bind Smc5 (mph1-Δ60). Remarkably, mph1-Δ60 supports normal growth and responses to DNA damaging agents, indicating that Smc5/6 does not simply restrain the recombinogenic activity of Mph1 via direct binding. These data as a whole highlight a role for Smc5/6 and Sgs1 in the resolution of Mph1-dependent HR intermediates.     

The N-terminal Domain of the Drosophila Mitochondrial Replicative DNA Helicase Contains an Iron-Sulfur Cluster and Binds DNA

The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome. It exhibits strong sequence homology with the bacteriophage T7 gene 4 protein primase-helicase (T7 gp4). Both proteins contain distinct N- and C-terminal domains separated by a flexible linker. The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain. Whereas the N-terminal domain in T7 gp4 contains a DNA primase activity, this function is lost in metazoan mtDNA helicase. Thus, although the functions of the C-terminal domain and the linker are partially understood, the role of the N-terminal region in the metazoan replicative mtDNA helicase remains elusive. Here, we show that the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro. The N-terminal domain binds the cluster through conserved cysteine residues (Cys⁶⁸, Cys⁷¹, Cys¹⁰², and Cys¹⁰⁵) that are responsible for coordinating zinc in T7 gp4. Moreover, we show that the N-terminal domain binds both single- and double-stranded DNA oligomers, with an apparent K_d of ∼120 nm. These findings suggest a possible role for the N-terminal domain of metazoan mtDNA helicase in recruiting and binding DNA at the replication fork.     

A257T linker region mutant of T7 helicase-primase protein is defective in DNA loading and rescued by T7 DNA polymerase

The helicase and primase activities of the hexameric ring-shaped T7 gp4 protein reside in two separate domains connected by a linker region. This linker region is part of the subunit interface between monomers, and point mutations in this region have deleterious effects on the helicase functions. One such linker region mutant, A257T, is analogous to the A359T mutant of the homologous human mitochondrial DNA helicase Twinkle, which is linked to diseases such as progressive external opthalmoplegia. Electron microscopy studies show that A257T gp4 is normal in forming rings with dTTP, but the rings do not assemble efficiently on the DNA. Therefore, A257T, unlike the WT gp4, does not preassemble on the unwinding DNA substrate with dTTP without Mg(II), and its DNA unwinding activity in ensemble assays is slow and limited by the DNA loading rate. Single molecule assays measured a 45 times slower rate of A257T loading on DNA compared with WT gp4. Interestingly, once loaded, A257T has almost WT-like translocation and DNA unwinding activities. Strikingly, A257T preassembles stably on the DNA in the presence of T7 DNA polymerase, which restores the ensemble unwinding activity of A257T to ～75% of WT, and the rescue does not require DNA synthesis. The DNA loading rate of A257T, however, remains slow even in the presence of the polymerase, which explains why A257T does not support T7 phage growth. Similar types of defects in the related human mitochondrial DNA helicase may be responsible for inefficient DNA replication leading to the disease states.     

The linker region between the helicase and primase domains of the bacteriophage T7 gene 4 protein is critical for hexamer formation.

The gene 4 protein of bacteriophage T7, a functional hexamer, comprises DNA helicase and primase activities. Both activities depend on the unidirectional movement of the protein along single-stranded DNA in a reaction coupled to the hydrolysis of dTTP. We have characterized dTTPase activity and hexamer formation for the full-length gene 4 protein (gp4) as well as for three carboxyl-terminal fragments starting at residues 219 (gp4-C219), 241 (gp4-C241), and 272 (gp4-C272). The region between residues 242 and 271, residing between the primase and helicase domains, is critical for oligomerization of the gene 4 protein. A functional TPase active site is dependent on oligomerization. During native gel electrophoresis, gp4, gp4-C219, and gp4-C241 migrate as oligomers, whereas gp4-C272 is monomeric. The steady-state k(cat) for dTTPase activity of gp4-C272 increases sharply with protein concentration, indicating that it forms oligomers only at high concentrations. gp4-C219 and gp4-C241 both form a stable complex with gp4, whereas gp4-C272 interacts only weakly with gp4. Measurements of surface plasmon resonance indicate that a monomer of T7 DNA polymerase binds to a dimer of gp4, gp4-C219, or gp4-C241 but to a monomer of gp4-C272. Like the homologous RecA and F(1)-ATPase proteins, the oligomerization domain of the gene 4 protein is adjacent to the amino terminus of the NTP-binding domain.