A partially functional DNA helicase II mutant defective in forming stable binary complexes with ATP and DNA. A role for helicase motif III

To address the functional significance of motif III in Escherichia coli DNA helicase II, the conserved aspartic acid at position 248 was changed to asparagine. UvrDD248N failed to form stable binary complexes with either DNA or ATP. However, UvrDD248N was capable of forming an active ternary complex when both ATP and single-stranded DNA were present. The DNA-stimulated ATPase activity of UvrDD248N was reduced relative to that of wild-type UvrD with no significant change in the apparent Km for ATP. The mutant protein also demonstrated a reduced DNA unwinding activity. The requirement for high concentrations of UvrDD248N to achieve unwinding of long duplex substrates likely reflects the reduced stability of various binary and ternary complexes that must exist in the catalytic cycle of a helicase. The data suggest that motif III may act as an interface between the ATP binding and DNA binding domains of a helicase. The uvrDD248N allele was also characterized in genetic assays. The D248N protein complemented the UV-sensitive phenotype of a uvrD deletion strain to levels nearly equivalent to wild-type helicase II. In contrast, the mutant protein only partially complemented the mutator phenotype. A correlation between the level of genetic complementation and the helicase activity of UvrDD248N is discussed.     

Human DNA helicase VIII: a DNA and RNA helicase corresponding to the G3BP protein, an element of the ras transduction pathway

Human DNA helicase VIII (HDH VIII) was isolated in the course of a systematic study of the DNA unwinding enzymes present in human cells. From a HeLa cell nuclear extract a protein with an Mrof 68 kDa in SDS-PAGE was isolated, characterised and micro-sequenced. The enzyme shows ATP- and Mg2+-dependent activity is not stimulated by RPA, prefers partially unwound 3'-tailed substrates and moves along the bound strand in the 5' to 3' direction. HDH VIII can also unwind partial RNA/DNA and RNA/RNA duplexes. Microsequencing of the polypeptide showed that this enzyme corresponds to G3BP, an element of the Ras pathway which binds specifically to the GTPase-activating protein. HDH VIII/G3BP is analogous to the heterogeneous nuclear ribonucleoproteins and contains a sequence rich in RGG boxes similar to the C-terminal domain of HDH IV/nucleolin, another DNA and RNA helicase.     

Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site

The 63 kDa gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain of the gene 4 protein is responsible for DNA-dependent NTP hydrolysis and for hexamer formation, whereas the N-terminal primase domain contains the zinc motif that is, in part, responsible for template-directed oligoribonucleotide synthesis. In the presence of beta, gamma-methylene dTTP, the protein forms a hexamer that surrounds and binds tightly to single-stranded DNA and consequently is unable to translocate to primase recognition sites, 5'-GTC-3', or to dissociate from the molecule to which it is bound. Nonetheless, in the presence of beta,gamma-methylene dTTP, it catalyzes the synthesis of pppAC dimers at primase sites on M13 DNA. When bound to single-stranded DNA in the presence of beta,gamma-methylene dTTP, the primase can function at recognition sites on the same molecule to which it is bound provided that a sufficient distance exists between the recognition site and the site to which it is bound. Furthermore, the primase bound to one DNA strand can function at a primase site located on a second DNA strand. The results indicate that the primase domain resides on the outside of the hexameric ring, a location that enables it to access sites distal to its site of binding.     

Mismatch-, MutS-, MutL-, and helicase II-dependent unwinding from the single-strand break of an incised heteroduplex

Escherichia coli MutS, MutL, and DNA helicase II are sufficient to initiate mismatch-dependent unwinding of an incised heteroduplex (Yamaguchi, M., Dao, V., and Modrich, P. (1998) J. Biol. Chem., 273, 9197-9201). We have studied unwinding of 6.4-kilobase circular G-T heteroduplexes that contain a single-strand incision, 808 base pairs 5' to the mismatch or 1023 base pairs 3' to the mispair as viewed along the shorter path between the two DNA sites. Unwinding of both substrates in the presence of MutS, MutL, DNA helicase II, and single-stranded DNA binding protein was mismatch-dependent and initiated at the single-strand break. Although unwinding occurred in both directions from the strand break, it was biased toward the shorter path linking the strand break and the mispair. MutS and MutL are thus sufficient to coordinate mismatch recognition to the orientation-dependent activation of helicase II unwinding at a single-strand break located a kilobase from the mispair.     

A hexameric helicase encircles one DNA strand and excludes the other during DNA unwinding

The bacteriophage T7 DNA helicase/primase (gene 4 protein) is a ring-like hexamer that encircles ssDNA and requires forked DNA to catalyze DNA unwinding. We report that optimal rates of unwinding of forked DNA require ssDNA tails of 55 nucleotides on the 5'-to-3' strand and 15 nucleotides on the 3'-to-5' strand. Surprisingly, streptavidin bound to a biotinylated 3'-end fully substitutes for the 3'-to-5' ssDNA tail. This suggests that excluding the 3'-to-5' DNA strand from the center of the helicase is an essential aspect of the mechanism of hexameric helicase-catalyzed DNA unwinding. We also report that streptavidin bound to a biotinylated dT within the 5'-to-3' strand of the duplexed region abolishes DNA unwinding; whereas, streptavidin bound to a biotinylated dT within the duplexed region of the other strand has no effect. These results unambiguously demonstrate that the T7 gene 4 protein is a 5'-to-3' helicase and imply that during DNA unwinding the 5'-to-3' strand transverses the center of the ring while the 3'-to-5' strand is excluded from the center of the ring. Implications for collisions between a helicase and other protein-DNA complexes are discussed.     

Herpes simplex virus type-1 single-strand DNA-binding protein (ICP8) enhances the ability of the viral DNA helicase-primase to unwind cisplatin-modified DNA

The herpes simplex virus type-1 UL5, UL8, and UL52 genes encode an essential heterotrimeric DNA helicase-primase that is responsible for concomitant DNA unwinding and primer synthesis at the viral DNA replication fork. The viral single-strand DNA-binding protein (ICP8) can stimulate DNA unwinding by the helicase-primase as a result of a physical interaction that is mediated by the UL8 subunit. In this study, we investigated the ability of the helicase-primase to unwind a fork-like substrate that contains an intrastrand d(GpG) DNA cross-link produced by the antitumor drug cisplatin. We also examined the ability of ICP8 to modulate the effect of the cisplatin lesion. The data show that the lesion inhibited the helicase-primase when located on the DNA strand along which it translocates. However, the lesion did not represent a permanent obstacle to its progression. In contrast, the adduct did not affect the helicase-primase when located on the opposite DNA strand. ICP8 specifically stimulated DNA unwinding by the helicase-primase. Coating concentrations of ICP8 were necessary for optimal unwinding of damaged DNA. Addition of competitor DNA to helicase reactions led to substantial reduction of DNA unwinding by the helicase-primase, suggesting that the enzyme is distributive. ICP8 did not abolish the competition, indicating that it did not stimulate the helicase by increasing its processivity. Rather, ICP8 may stimulate DNA unwinding and enable bypass of cisplatin damaged DNA by recruiting the helicase-primase to the DNA.     

The replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication

Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase alpha-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.     

Helicase motifs V and VI of the Escherichia coli UvrB protein of the UvrABC endonuclease are essential for the formation of the preincision complex

The UvrB protein is a subunit of the UvrABC endonuclease which is involved in the repair of a large variety of DNA lesions. We have 91 isolated random uvrB mutants which are impaired in the repair of UV-damage in vivo. These mutants were classified on the basis of the ability to form normal levels of protein and the position of the mutations in the gene. The amino acid substitutions in the N-terminal part or in the C-terminal part of the UvrB protein are exclusively found in the conserved boxes of the so-called "helicase motifs" present in these parts of the protein, indicating that these motifs are essential for UvrB function. The proteins of four C-terminal mutants were purified: two mutants in motif V (E514K and G509S), one mutant in motif VI (R544H) and a double mutant in both motifs (E514K + R541H). In vitro experiments with these mutant proteins show that the helicase motifs V and VI are involved in the induction of ATP hydrolysis in the presence of (damaged) DNA and in the strand-displacement activity of the UvrA2B complex as is observed in a helicase assay. Furthermore, our results suggest that this strand-displacement activity is correlated to a local unwinding, which seems to be used to form the UvrB-DNA preincision complex.     

Asymmetric interactions of hexameric bacteriophage T7 DNA helicase with the 5'- and 3'-tails of the forked DNA substrate

Bacteriophage T7 DNA helicase requires two noncomplementary single-stranded DNA (ssDNA) tails next to a double-stranded DNA (dsDNA) region to initiate DNA unwinding. The interactions of the helicase with the DNA were investigated using a series of forked DNAs. Our results show that the helicase interacts asymmetrically with the two tails of the forked DNA. When the helicase was preassembled on the forked DNA before the start of unwinding, a DNA with 15-nucleotide (nt) 3'-tail and 35-nt 5'-tail was unwound with optimal rates close to 60 base pairs/s at 18 degrees C. When the helicase was not preassembled on the DNA, a >65-nt long 5'-tail was required for maximal unwinding rates of 12 base pairs/s. We show that the helicase interacts specifically with the ssDNA region and maintains contact with both ssDNA strands during DNA unwinding, since conversion of the two ssDNA tails to dsDNA structures greatly inhibited unwinding, and the helicase was unable to unwind past a nick in the dsDNA region. These studies have provided new insights into the mechanism of DNA unwinding. We propose an exclusion model of DNA unwinding in which T7 helicase hexamer interacts mainly with the ssDNA strands during DNA unwinding, encircling the 5'-strand and excluding the 3'-strand from the hole.     

The Bloom's syndrome helicase unwinds G4 DNA

BLM, the gene that is defective in Bloom's syndrome, encodes a protein homologous to RecQ subfamily helicases that functions as a 3'-5' DNA helicase in vitro. We now report that the BLM helicase can unwind G4 DNA. The BLM G4 DNA unwinding activity is ATP-dependent and requires a short 3' region of single-stranded DNA. Strikingly, G4 DNA is a preferred substrate of the BLM helicase, as measured both by efficiency of unwinding and by competition. These results suggest that G4 DNA may be a natural substrate of BLM in vivo and that the failure to unwind G4 DNA may cause the genomic instability and increased frequency of sister chromatid exchange characteristic of Bloom's syndrome.     

Nucleotide sequence analysis of the CDNA for rat DNA topoisomerase II

CDNA clones encoding the rat DNA topoisomerase II were isolated from rat testis CDNA library using a DNA probe synthesized by two sequential nested PCRs. The nucleotide sequence of the entire coding region and its deduced 1526 amino acid sequence showed that 80% nucleotides and 89% amino acids were identical with human HeLa DNA topoisomerase II gene (hTOP2). Approximately 1100 amino acids at the N-terminus shows 96.5% sequence identity, but C-terminus has only 65% homology. Rat DNA topoisomerase II gene (rTOP2) contains three functional domains responsible for ATPase activity, break-reunion activity, and complex stability and DNA binding activity like other eukaryotic TOP2. It also contains two putative nuclear targeting sequences and a leucine zipper motif and has highly charged species specific sequences at the C-terminus.     

Regulatory implications of protein assemblies at the gamma origin of plasmid R6K - a review

Recognition of the replication origin (ori) by initiator protein is a recurring theme for the regulated initiation of DNA replication in diverse biological systems. The objective of the work reviewed here is to understand the initiation process focusing specifically on the gamma-ori of the antibiotic-resistance plasmid R6K. The control of gamma-ori copy number is determined by both plasmid-encoded and host-encoded factors. The two central regulatory elements of the plasmid are a multifunctional initiator protein pi, and sequence-related DNA target sites, the inverted half-repeats (IRs) and the direct repeats (DRs). The replication activator and inhibitor activities of pi seem to be at least partially distributed between two naturally occurring pi polypeptides (designated by their molecular weights pi35.0 and pi30.5). Regulatory variants of pi with altered states of oligomerization in nucleoprotein complexes with DRs and IRs have been isolated. The properties of these mutants laid the foundation for our model of pi protein activity which proposes that different protein surfaces are required for the formation of functionally distinct complexes of pi with DRs and IRs. These mutants also suggest that pi polypeptides have a modular structure; the C-terminus contains the DNA-binding domain while the N-terminus controls protein oligomerization. Additionally, pi35.0 binds to a novel DNA sequence in the A+T-rich segment of gamma-ori. This binding site is at or near the site from which synthesis of the leading strand begins.     

Deletion analysis of MotA and MotB, components of the force-generating unit in the flagellar motor of Salmonella

MotA and MotB are cytoplasmic membrane proteins that form the force-generating unit of the flagellar motor in Salmonella typhimurium and many other bacteria. Many missense mutations in both proteins are known to cause slow motor rotation (slow-motile phenotype) or no rotation at all (non-motile or paralysed phenotype). However, large stretches of sequence in the cytoplasmic regions of MotA and in the periplasmic region of MotB have failed to yield these types of mutations. In this study, we have investigated the effect of a series of 10-amino-acid deletions in these phenotypically silent regions. In the case of MotA, we found that only the C-terminal 5 amino acids were completely dispensable; an adjacent 10 amino acids were partially dispensable. In the cytoplasmic loop region of MotA, deletions made the protein unstable. For MotB, we found that two large segments of the periplasmic region were dispensable: the results with individual deletions showed that the first consisted of six deletions between the sole transmembrane span and the peptidoglycan binding motif, whereas the second consisted of four deletions at the C-terminus. We also found that deletions in the MotB cytoplasmic region at the N-terminus impaired motility but did not abolish it. Further investigations in MotB were carried out by combining dispensable deletion segments. The most extreme version of MotB that still retained some degree of function lacked a total of 99 amino acids in the periplasmic region, beginning immediately after the transmembrane span. These results indicate that the deleted regions in the MotA cytoplasmic loop region are essential for stability; they may or may not be directly involved in torque generation. Part of the MotA C-terminal cytoplasmic region is not essential for torque generation. MotB can be divided into three regions: an N-terminal region of about 30 amino acids in the cytoplasm, a transmembrane span and about 260 amino acids in the periplasm, including a peptidoglycan binding motif. In the periplasmic region, we suggest that the first of the two dispensable stretches in MotB may comprise part of a linker between the transmembrane span of MotB and its attachment point to the peptidoglycan layer, and that the length or specific sequence of much of that linker sequence is not critical. About 40 residues at the C-terminus are also unimportant.     

A central region of Ku80 mediates interaction with Ku70 in vivo

Ku, the DNA binding component of DNA-dependent protein kinase (DNA-PK), is a heterodimer composed of 70 and 86 kDa subunits, known as Ku70 and Ku80 respectively . Defects in DNA-PK subunits have been shown to result in a reduced capacity to repair DNA double-strand breaks. Assembly of the Ku heterodimer is required to obtain DNA end binding activity and association of the DNA-PK catalytic subunit. The regions of the Ku subunits responsible for heterodimerization have not been clearly defined in vivo . A previous study has suggested that the C-terminus of Ku80 is required for interaction with Ku70. Here we examine Ku subunit interaction using N- and C-terminal Ku80 deletions in a GAL4-based two-hybrid system and an independent mammalian in vivo system. Our two-hybrid study suggests that the central region of Ku80, not its C-terminus, is capable of mediating interaction with Ku70. To determine if this region mediates interaction with Ku70 in mammalian cells we transfected xrs-6 cells, which lack endogenous Ku80, with epitope-tagged Ku80 deletions carrying a nuclear localization signal. Immunoprecipitation from transfected cell extracts revealed that the central domain identified by the GAL4 two-hybrid studies stabilizes and co-immunoprecipitates with endogenous xrs-6 Ku70. The central interaction domain maps to the internally deleted regions of Ku80 in the mutant cell lines XR-V9B and XR-V15B. These findings indicate that the internally deleted Ku80 mutations carried in these cell lines are incapable of heterodimerization with Ku70.     

Recognition of diverse sequences by class I zinc fingers: asymmetries and indirect effects on specificity in the interaction between CF2II and A+T-rich elements

The Drosophila CF2II protein, which contains zinc fingers of the Cys2His2 type and recognizes an A+T-rich sequence, behaves in cell culture as an activator of a reporter chloramphenicol acetyltransferase gene. This activity depends on C-terminal but not N-terminal zinc fingers, as does in vitro DNA binding. By site-specific mutagenesis and binding site selection, we define the critical amino acid-base interactions. Mutations of single amino acid residues at the leading edge of the recognition helix are rarely neutral: many result in a slight change in affinity for the ideal DNA target site; some cause major loss of affinity; and others change specificity for as many as two bases in the target site. Compared to zinc fingers that recognize G+C-rich DNA, CF2II fingers appear to bind to A+T-rich DNA in a generally similar manner, but with additional flexibility and amino acid-base interactions. The results illustrate how zinc fingers may be evolving to recognize an unusually diverse set of DNA sequences.     

Crystal structure of the site-specific recombinase, XerD

The structure of the site-specific recombinase, XerD, that functions in circular chromosome separation, has been solved at 2.5 A resolution and reveals that the protein comprises two domains. The C-terminal domain contains two conserved sequence motifs that are located in similar positions in the structures of XerD, lambda and HP1 integrases. However, the extreme C-terminal regions of the three proteins, containing the active site tyrosine, are very different. In XerD, the arrangement of active site residues supports a cis cleavage mechanism. Biochemical evidence for DNA bending is encompassed in a model that accommodates extensive biochemical and genetic data, and in which the DNA is wrapped around an alpha-helix in a manner similar to that observed for CAP complexed with DNA.     

Escherichia coli RuvBL268S: a mutant RuvB protein that exhibits wild-type activities in vitro but confers a UV-sensitive ruv phenotype in vivo

The RuvABC proteins of Escherichia coli process recombination intermediates during genetic recombination and DNA repair. RuvA and RuvB promote branch migration of Holliday junctions, a process that extends heteroduplex DNA. Together with RuvC, they form a RuvABC complex capable of Holliday junction resolution. Branch migration by RuvAB is mediated by RuvB, a hexameric ring protein that acts as an ATP-driven molecular pump. To gain insight into the mechanism of branch migration, random mutations were introduced into the ruvB gene by PCR and a collection of mutant alleles were obtained. Mutation of leucine 268 to serine resulted in a severe UV-sensitive phenotype, characteristic of a ruv defect. Here, we report a biochemical analysis of the mutant protein RuvBL268S. Unexpectedly, the purified protein is fully active in vitro with regard to its ATPase, DNA binding and DNA unwinding activities. It also promotes efficient branch migration in combination with RuvA, and forms functional RuvABC-Holliday junction resolvase complexes. These results indicate that RuvB may perform some additional, and as yet undefined, function that is necessary for cell survival after UV-irradiation.