NORMAL GENOMIC

• Ha JY et al.
• The recombination-associated protein RdgC adopts a novel toroidalarchitecture for DNA binding.
• Nucleic Acids Res. 2007; 35: 2671-81
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• RecA plays a central role in the nonmutagenic repair of stalledreplication forks in bacteria. RdgC, a recombination-associatedDNA-binding protein, is a potential negative regulator of RecA function.Here, we have determined the crystal structure of RdgC from Pseudomonasaeruginosa. The J-shaped monomer has a unique fold and can be divided intothree structural domains: tip domain, center domain and base domain. Twosuch monomers dimerize to form a ring-shaped molecule of approximate2-fold symmetry. Of the two inter-subunit interfaces within the dimer, oneinterface ('interface A') between tip/center domains is more nonpolar thanthe other ('interface B') between base domains. The structure allows us topropose that the RdgC dimer binds dsDNA through the central hole ofapproximately 30 A diameter. The proposed model is supported by ourDNA-binding assays coupled with mutagenesis, which indicate that theconserved positively charged residues on the protein surface around thecentral hole play important roles in DNA binding. The novel ring-shapedarchitecture of the RdgC dimer has significant implications for its rolein homologous recombination.

• Kongsuwan K, Josh P, Picault MJ, Wijffels G, Dalrymple B
• The plasmid RK2 replication initiator protein (TrfA) binds to the slidingclamp beta subunit of DNA polymerase III: implication for the toxicity ofa peptide derived from the amino-terminal portion of 33-kilodalton TrfA.
• J Bacteriol. 2006; 188: 5501-9
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• The broad-host-range plasmid RK2 is capable of replication and stablemaintenance within a wide range of gram-negative bacterial hosts. Itencodes the essential replication initiation protein TrfA, which binds tothe host initiation protein, DnaA, at the plasmid origin of replication(oriV). There are two versions of the TrfA protein, 44 and 33 kDa,resulting from alternate in-frame translational starts. We have shown thatthe smaller protein, TrfA-33, and its 64-residue amino-terminal peptide(designated T1) physically interact with the Escherichia coli beta slidingclamp (beta(2)). This interaction appears to be mediated through a QLSLFpeptide motif located near the amino-terminal end of TrfA-33 and T1, whichis identical to the previously described eubacterial clamp-bindingconsensus motif. T1 forms a stable complex with beta(2) and was found toinhibit plasmid RK2 replication in vitro. This specific interactionbetween T1 and beta(2) and the ability of T1 to block DNA replication haveimplications for the previously reported cell lethality caused byoverproduction of T1. The toxicity of T1 was suppressed when wild-type T1was replaced with mutant T1, carrying an LF deletion in the beta-bindingmotif. Previously, T1 toxicity has been shown to be suppressed by Hda, anintermediate regulatory protein which helps prevent over-initiation in E.coli through its interaction with the initiator protein, DnaA, andbeta(2). Our results support a model in which T1 toxicity is caused by T1binding to beta(2), especially when T1 is overexpressed, preventingbeta(2) from interacting with host replication proteins such as Hda duringthe early events of chromosome replication.

• Sanders CM, Stenlund A
• Recruitment and loading of the E1 initiator protein: an ATP-dependentprocess catalysed by a transcription factor.
• EMBO J. 1998; 17: 7044-55
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• Initiation of DNA replication critically depends on ori recognition aswell as on catalytic activities of the initiator complex. For replicationof papillomaviruses the catalytic activities for initiation are providedby the E1 protein. Here, we show that the transcription factor E2 acts toassemble E1 into a complex active for ori distortion in two steps. First,cooperative DNA binding of E1 and E2 generates a sequence-specific orirecognition complex. In the second ATP-dependent step, E2 is displaced andadditional E1 molecules are incorporated. The net result is a finalcomplex with low sequence specificity deposited onto a specific sequencein the DNA. This may be a general strategy to accomplish specificpositioning of protein complexes with low sequence specificity.

• Berthomme H, Monahan SJ, Parris DS, Jacquemont B, Epstein AL
• Cloning, sequencing, and functional characterization of the two subunitsof the pseudorabies virus DNA polymerase holoenzyme: evidence forspecificity of interaction.
• J Virol. 1995; 69: 2811-8
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• The pseudorabies virus (PRV) genes encoding the two subunits of the DNApolymerase were located on the genome by hybridization to their herpessimplex virus type 1 (HSV-1) homologs, pol and UL42, and subsequently weresequenced. Like the HSV-1 homologs, in vitro translation products of thePRV gene encoding the catalytic subunit (pol) possessed activity in theabsence of the Pol accessory protein (PAP). However, the PRV PAPstimulated the activity of Pol fourfold in the presence of 150 mM KCl,using an activated calf thymus DNA template. The stimulation of Polactivity by PAP under high-salt conditions and the inhibition of Polactivity by PAP when assayed in low salt (0 mM KCl) together were used todetermine the specificity with which PAP interacted with Pol. Despitefunctional similarity, HSV-1 UL42 and PRV PAP could neither stimulate thenoncognate Pols at high salt nor inhibit them at low salt. Furthermore, aPRV Pol mutant lacking the 30 C-terminal amino acids retained basal Polactivity but could be neither stimulated nor inhibited by the PRV PAP.Sequence comparisons of the Pol proteins of the alphaherpesviruses reveala conserved domain in the C terminus which terminates immediately beforethe last 41 residues of both PRV and HSV-1 proteins. These resultsindicate that the ability and specificity for interaction of the PRV Polwith PAP most likely resides predominantly in the extreme Pol C terminus.

• Podust LM, Podust VN, Floth C, Hubscher U
• Assembly of DNA polymerase delta and epsilon holoenzymes depends on thegeometry of the DNA template.
• Nucleic Acids Res. 1994; 22: 2970-5
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• To study in details the assembly of DNA polymerases delta and epsilonholoenzymes a circular double-stranded DNA template containing a gap of 45nucleotides was constructed. Both replication factor C and proliferatingcell nuclear antigen were absolutely required and sufficient for assemblyof DNA polymerase delta holoenzyme complex on DNA. On such a circular DNAsubstrate replication protein A (or E. coli single-strand DNA bindingprotein) was neither required for assembly of DNA polymerase deltaholoenzyme complex nor for the gap-filling reaction. A circular structureof the DNA substrate was found to be absolutely critical for the abilityof auxiliary proteins to interact with DNA polymerases. The linearizationof the circular DNA template resulted in three dramatic effects: (i) DNAsynthesis by DNA polymerase delta holoenzyme was abolished, (ii) theinhibition effect of replication factor C and proliferating cell nuclearantigen on DNA polymerase alpha was relieved and (iii) DNA polymeraseepsilon could not form any longer a holoenzyme with replication factor Cand proliferating cell nuclear antigen. The comparison of the effect ofreplication factor C and proliferating cell nuclear antigen on DNApolymerases alpha, delta and epsilon indicated that the auxiliary proteinsappear to form a mobile clamp, which can easily slide alongdouble-stranded DNA.