Dda, one of three helicases encoded by bacteriophage T4, has been well- characterized biochemically but its biological role remains unclear. It is thought to be involved in origin-dependent replication, recombination-dependent replication, anti- recombination, recombination repair, as well as in replication fork progression past template-bound nucleosomes and RNA polymerase. One of the proteins that most strongly interacts with Dda, Gp32, is the only single-stranded DNA binding protein (SSB) encoded by T4, is essential for DNA replication, recombination, and repair. Previous studies have shown that Gp32 is essential for Dda stimulation of replication fork progression. Our studies show that interactions between Dda and Gp32 play a critical role in regulating replication fork restart during recombination repair. When the leading strand polymerase stalls at a site of ssDNA damage and the lagging strand machinery continues, Gp32 binds the resulting ssDNA gap ahead of the stalled leading strand polymerase. We found that a Gp32 cluster on leading strand ssDNA blocks Dda loading on the lagging strand ssDNA, blocks stimulation of fork progression by Dda, and stimulates Dda to displace the stalled polymerase and the 3' end of the daughter strand. This unwinding generates conditions necessary for polymerase template switching in order to regress the DNA damage-stalled replication fork. Helicase trafficking by Gp32 could play a role in preventing premature fork progression until the events required for error-free translesion DNA synthesis have taken place. Interestingly, we found that Dda helicase activity is strongly stimulated by the N-terminal deletion mutant Gp32-B, suggesting the N-terminal truncation to generate Gp32-B reveals a cryptic helicase stimulatory activity of Gp32 that may be revealed in the context of a moving polymerase, or through direct interactions of Gp32 with other replisome components. Additionally, our findings support a role for Dda-Gp32 interactions in double strand break (DSB) repair by homology-directed repair (HDR), which relies on homologous recombination and the formation of a displacement loop (D-loop) that can initiate DNA synthesis. We examined the D-loop unwinding activity of Dda, Gp41, and UvsW, the D-loop strand extension activity of Gp43 polymerase, and the effect of the helicases and their modulators on D-loop extension. Dda and UvsW, but not Gp41, catalyze D-loop invading strand by DNA unwinding. The relationship between Dda and Gp43 was modulated by the presence of Gp32. Dda D-loop unwinding competes with D- loop extension by Gp43 only in the presence of Gp32, resulting in a decreased frequency of invading strand extension when all three proteins are present. These data suggest Dda functions as an antirecombinase and negatively regulates the replicative extension of D- loops. Invading strand extension is observed in the presence of Dda, indicating that invading strand extension and unwinding can occur in a coordinated manner. The result is a translocating D-loop, called bubble migration synthesis, a hallmark of break-induced repair (BIR) and synthesis dependent strand annealing (SDSA). Gp41 did not unwind D- loops studied and may serve as a secondary helicase loaded subsequent to D-loop processing by Dda. Dda is proposed to be a mixed function helicase that can work both as an antirecombinase and to promote recombination-dependent DNA synthesis, consistent with the notion that Dda stimulates branch migration. These results have implications on the repair of ssDNA damage, DSB repair, and replication fork regulation, which are highly conserved processes sustained in all organisms.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25357号 / 農博第2623号 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 井上 和生, 教授 谷 史人 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing. Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension. The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results. Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses. Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results. <b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis

<p>The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing.</p><p>Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension.</p><p>The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results.</p><p>Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses.</p><p>Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results.</p><p><b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis</p>

For survival and successful propagation, every organism has to maintain the genomic integrity of the cell. The information content, in the form of nucleotide bases, is constantly threatened by endogenous agents and environmental pollutants. In particular, pathogenic mycobacteria are constantly exposed to DNA-damaging assaults such as reactive oxygen species (ROS) and reactive nitrogen intermediate (RNI), in their habitat which is inside host macrophage. In addition, the genome of Mycobacterium tuberculosis makes it more susceptible for guanine oxidation and cytosine deamination as it is G-C rich. Therefore DNA repair mechanisms are extremely important for the mycobacterium. An important enzyme involved in DNA repair is uracil-DNA glycosylase (Ung). To access the genomic information, during repair as well as DNA replication and recombination, dsDNA must unwind to form single stranded (ss) intermediates. ssDNA is more prone to chemical and nuclease attacks that can produce breaks or lesions and can also inappropriately self associate. In order to preserve ssDNA intermediates, cells have evolved a specialized class of ssDNA-binding proteins (SSB) that associate with ssDNA with high affinity. As part of a major programme on mycobacterial proteins in this laboratory, structural studies on mycobacterial uracil-DNA glycosylase (Ung) and single-stranded DNA binding protein (SSB) have been carried out. The structures were solved using the well-established techniques of protein X-ray crystallography. The hanging drop vapour diffusion and microbatch methods were used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator. The data were processed using the HKL program suite. The structures were solved by the molecular replacement method using the program PHASER and AMoRe. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using COOT. PROCHECK, ALIGN, INSIGHT and NACCESS were used for structure validation and analysis of the refined structures. MD simulations were performed using the software package GROMACS v 3.3.1. Uracil-DNA glycosylase (UNG), a repair enzyme involved in the excision of uracil from DNA, from mycobacteria differs from UNGs from other sources, particularly in the sequence in the catalytically important loops. The structure of the enzyme from Mycobacterium tuberculosis (MtUng) in complex with a proteinaceous inhibitor (Ugi) has been determined by X-ray analysis of a crystal containing seven crystallographically independent copies of the complex. This structure provides the first geometric characterization of a mycobacterial UNG. A comparison of the structure with those of other UNG proteins of known structure shows that a central core region of the molecule is relatively invariant in structure and sequence, while the N- and C-terminal tails exhibit high variability. The tails are probably important in folding and stability. The mycobacterial enzyme exhibits differences in UNG-Ugi interactions compared with those involving UNG from other sources. The MtUng-DNA complex modelled on the basis of the known structure of the complex involving the human enzyme indicates a domain closure in the enzyme when binding to DNA. The binding involves a larger burial of surface area than is observed in binding by human UNG. The DNA-binding site of MtUng is characterized by the presence of a higher proportion of arginyl residues than is found in the binding site of any other UNG of known structure. In addition to the electrostatic effects produced by the arginyl residues, the hydrogen bonds in which they are involved compensate for the loss of some interactions arising from changes in amino-acid residues, particularly in the catalytic loops. The results arising from the present investigation represent unique features of the structure and interaction of mycobacterial Ungs. To gain further insights, the structure of Mycobacterium tuberculosis Ung (MtUng) in its free form was also determined. Comparison with appropriate structures indicate that the two domain enzyme slightly closes up when binding to DNA while it slightly opens up when binding to its proteinaceous inhibitor Ugi. The structural changes on complexation in the catalytic loops reflect the special features of their structure in the mycobacterial protein. A comparative analysis of available sequences of the enzyme from different sources indicates high conservation of amino acid residues in the catalytic loops. The uracil binding pocket in the structure is occupied by a citrate ion. The interactions of the citrate ion with the protein mimic those of uracil in addition to providing insights into other possible interactions that inhibitors could be involved in. SSB is an essential accessory protein required during DNA replication, repair and recombination, and various other DNA transactions. Eubacteral single stranded DNA binding (SSB) proteins constitute an extensively studied family of proteins. The variability in the quaternary association in these tetrameric proteins was first demonstrated through the X-ray analysis of the crystal structure of Mycobacterium tuberculosis SSB (MtSSB) and Mycobacterium smegmatis (MsSSB) in this laboratory. Subsequent studies on these proteins elsewhere have further explored this variability, but attention was solely concentrated on the variability in the relative orientation of the two dimers that constitute the tetramer. Furthermore, the effect of this variability on the properties of the tetrameric molecule was not adequately addressed. In order to further explore this variability and strengthen structural information on mycobacterial SSBs in particular, and on SSB proteins in general, the crystal structures of two forms of Mycobacterium leprae single stranded DNA-binding protein (MlSSB) has been determined. Comparison of the structures with other eubacterial SSB structures indicates considerable variation in their quaternary association although the DNA binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but it appears to correlate well with variation in protein stability. Molecular dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype E. coli SSB and the individual subunits of both the proteins. The X-ray studies and molecular dynamics simulations together yield information on the relatively rigid and flexible regions of the molecule and the effect of oligomerization on flexibility. The simulations provide insights into the changes in the subunit structure on oligomerization. They also provide insights into the stability and time evolution of the hydrogen bonds/water-bridges that connect two pairs of monomers in the tetramer. In continuation of our effort to understand structure-function relationships of mycobacterial SSBs, the structure of MsSSB complexed with a 31-mer polydeoxy-cytidine single stranded DNA (ssDNA) was determined. The mode of ssDNA binding in the MsSSB is different from the modes in the known structures of similar complexes of the proteins from E. coli (EcSSB) and Helicobacter pylori (HpSSB). The modes in the EcSSB and HpSSB also exhibit considerable differences between them. A comparison of the three structures reveals the promiscuity of DNA-binding to SSBs from different species in terms of symmetry and the path followed by the bound DNA chain. It also reveals commonalities within the diversity. The regions of the protein molecule involved in DNA-binding and the nature of the residues which interact with the DNA, exhibit substantial similarities. The regions which exhibit similarities are on the central core of the subunit which is unaffected by tetramerisation. The variable features of DNA binding are associated with the periphery of the subunit, which is involved in oligomerization. Thus, there is some correlation between variability in DNA-binding and the known variability in tetrameric association in SSBs. In addition to the work on Ung and SSB, the author was involved in X-ray studies on crystals of horse methemoglobin at different levels of hydration, which is described in the Appendix of the thesis. The crystal structure of high-salt horse methaemoglobin has been determined at environmental relative humidities (r.h.) of 88, 79, 75 and 66%. The molecule is in the R state in the native and the r.h. 88% crystals. At r.h.79% the molecule appears to move towards the R2 state. The crystal structure at r.h.66% is similar, but not identical, to that at r.h.75%. Thus variation in hydration leads to variation in the quaternary structure. Furthermore, partial dehydration appears to shift the structure from the R state to the R2 state. This observation is in agreement with the earlier conclusion that the changes in protein structure that accompany partial dehydration are similar to those that occur during protein action. A part of the work presented in the thesis has been reported in the following publications. 1. Singh, P., Talawar, R.K., Krishna, P.D., Varshney, U. & Vijayan, M. (2006). Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor. Acta Crystallogr. F62, 1231-1234. 2. Kaushal, P.S., Talawar, R.K., Krishna, P.D., Varshney, U. & Vijayan, M. (2008). Unique features of the structure and interactions of mycobacterial uracil-DNA glycosylase: structure of a complex of the Mycobacterium tuberculosis enzyme in comparison with those from other sources. Acta Crystallogr. D64, 551-560. 3. Kaushal, P.S., Sankaranarayanan, R. & Vijayan, M. (2008). Water-mediated variability in the structure of relaxed-state haemoglobin. Acta Crystallogr. F64, 463-469.

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