Atomic Force Microscopy Studies of Eukaryotic Clamp/Clamp Loader Complex and Mismatch Repair Initiation Complex

As an advanced single molecule technique, atomic force microscopy (AFM) is a powerful and versatile tool for high resolution surface imaging and probing physical properties of soft, nonconductive bio-materials in vitro. Imaging of protein-protein and protein-DNA complexes provides structural and conformational information about the interactions of these biomolecular assemblies. In this study, we have used AFM to examine two different protein complexes: the eukaryotic RFC complex function in loading PCNA clamp onto different DNA substrate and eukaryotic MutS homologs function in the initiation of DNA mismatch repair (MMR). In the study of clamp loader RFC complex, we investigated the effect of nucleotide cofactors on the oligomerization states of RFC interacting with PCNA and DNA substrate. We observed that ATP binding induces a conformational change of RFC and that ATP hydrolysis causes RFC dissociation into small subcomplexes. However, PCNA inhibits the ATP-induced disassembly of RFC. Intriguingly, we found in the presence of ATP, some of the RFC subunits are ejected from DNA substrate, leaving RFC subcomplex bound to the DNA, and it appears that these subcomplexes form stable interaction with PCNA on the DNA. We proposed that this DNA-bound RFC subcomplex tethers PCNA ring at the single strand/double strand junction of primer-template DNA or nick DNA. We further suggest that dissociation of RFC subcomplex from PCNA and DNA substrate is promoted by downstream PCNA-interacting proteins, such as DNA polymerase. In addition to these insights into the complicated potential loading mechanism of PCNA, we observed other RFC-DNA complexes such as RFC-DNA filaments with nicked DNA without nucleotide cofactor and RFC-DNA spider-like complexes containing multiple RFCs and DNAs in the presence of ATP. Although we do not know the physiological role, if any, of such RFC-DNA complexes, these complexes suggest RFC can possess other functions besides as clamp loader, such as helicase. In the study of MMR initiation complexes, eukaryotic MutS homologs (MutSalpha and MutSbeta), we found, unlike their prokaryotic homologs, eukaryiotic MutS homologs bind different DNA substrates with similar conformation. MutSalpha and MutSbeta both exhibits weak binding specificity to their specific DNA substrates, which makes it more complicated to analyze their specific complexes. However, it appears that eukaryotic MutS homologs do not recognize mismatched bases simply depending on the formation of unbent complexes as seen in the prokaryotic MutS. It is possible they employ other high class mechanism in which the event of recognition of different mismatched DNA substrates happens downstream of mismatch binding

Electrostatic Switching Controls Channel Dynamics of the Sensor Protein VirB10 in A. tumefaciens Type IV Secretion System

Type IV secretion systems are large nanomachines assembled across the bacterial cell envelope for effector translocation and conjugation. VirB10 traverses the inner and outer membranes, sensing cellular signals for coordinating the conformational switch for pilus biogenesis and/or secretion. Mutations uncoupling secretion from pilus biogenesis were identified in Agrobacterium tumefaciens VirB10 including a gating defect mutation G272R that made VirB10 unresponsive to intracellular ATP, causing unregulated secretion of VirE2 in a contact-independent manner. Comparative long-timescale molecular dynamics of the wild type and G272R mutant of the A. tumefaciens VirB10_{CTD} tetradecamer reveals how the G272R mutation locks the oligomer in a rigid conformation by swapping the ionic interactions between the loops from the β-barrel close to the inner leaflet of the outer membrane. This electrostatic switching changes the allosteric communication pathway from the extracellular loop to the base of the barrel, suggesting that the local conformational dynamics in the loops can gate information across VirB10

Meiotic Recombination: The Essence of Heredity.

The study of homologous recombination has its historical roots in meiosis. In this context, recombination occurs as a programmed event that culminates in the formation of crossovers, which are essential for accurate chromosome segregation and create new combinations of parental alleles. Thus, meiotic recombination underlies both the independent assortment of parental chromosomes and genetic linkage. This review highlights the features of meiotic recombination that distinguish it from recombinational repair in somatic cells, and how the molecular processes of meiotic recombination are embedded and interdependent with the chromosome structures that characterize meiotic prophase. A more in-depth review presents our understanding of how crossover and noncrossover pathways of meiotic recombination are differentiated and regulated. The final section of this review summarizes the studies that have defined defective recombination as a leading cause of pregnancy loss and congenital disease in humans

Genetic analysis of two structure-specific endonucleases Hef and Fen1 in archaeon Haloferax volcanii

Nucleotide excision repair (NER) is a versatile pathway of DNA repair that deals with a variety of DNA lesions, such as UV-induced DNA damage and interstrand crosslinks. In bacteria, the UvrABC system carries out NER. In human cells, XPF and XPG are two structure-specific endonucleases that act in NER. XPF is responsible for a 5' incision at the DNA lesion and XPG carries out the 3' incision. In Archaea, the third domain of life, most species have homologues of some eukaryal NER proteins. Interestingly, Haloferax volcanii encodes homologues of both the eukaryotic NER genes (XPF, XPG, XPB and XPD) and bacterial NER genes (uvrA, uvrB, uvrC and uvrD). In this study, the function of XPG, XPF and UvrA in H. volcanii is investigated. XPG is related to FEN1, a structure-specific 5' flap endonuclease that acts in Okazaki fragment maturation. H. volcanii has a single gene homologous to both XPG and FEN1. The helicase/nuclease hef gene in H. volcanii is the archaeal homologue of human XPF, but also shows homology to Mus81 and FANCM. Mus81 has been found to resolve joint molecules in yeast, while FANCM is required for the repair of interstrand crosslinks in vertebrates. The uvrA gene in H. volcanii is the archaeal homologue of bacterial uvrA, which encodes a protein that plays a vital role in NER at the DNA damage recognition step. This study demonstrates that in H. volcanii, UvrA is involved in the major pathway for repair of UV induced DNA damage. By contrast, Hef and UvrA are involved in two different pathways for the repair of mitomycin C induced DNA crosslinks. Fen1 and Hef have overlapping functions for the repair of DNA cross-links, but not oxidative damage. We also obtain a spontaneous suppressor sfnA, which can suppress the slow growth and MMC sensitivity, but not the UV sensitivity of fen1 deletion mutants. Using plasmid assays, it has been shown that the hef deletion mutant is deficient in accurate end-joining and homologous recombination, including both crossover and non-crossover recombination. In contrast, Fen1 has no significant role in accurate end-joining, but Fen1 may regulate the ratio of non-crossover recombination to crossover recombination