Human Rad52 binding renders ssDNA unfolded: image and contour length analyses by Atomic Force Microscopy

Atomic force microscopy imaging has been used to study the changes associated with human Rad52 (HsRad52) protein in solution, in dried state as well as following ssDNA (linear and circular) binding. In the dried state, the free protein exists predominantly as a characteristic panoply of novel trifoliate forms. However, in solution, the level of trifoliates diminishes significantly. Height analyses of either form reveal two categories: smaller (~ 3-5 nm) and larger ((~ 10-12 nm) particles, perhaps related to sub-heptameric and heptameric forms respectively. Interestingly, binding of the protein to linear ssDNA smoothly extends and unfolds the naked DNA. Contour length measurements performed on several individual circular ssDNA/nucleoprotein complexes reveal marked (about threefold) extension of naked ssDNA, following HsRad52 binding. We speculate that the alignment of HsRad52 on ssDNA into a smoothly extended and unfolded strand from that of highly compact morphology of naked ssDNA, may have bearing on the recombination function of HsRad52 protein

Effect of DNA sequence and nucleotide cofactors on hRad51 binding to ssDNA: role of hRad52 in recruitment

hRad51 binding to ssDNA is significantly lowered in the presence of a nucleotide cofactor ATP/ADP/ATPγS. In these conditions, presence of trace amounts of hRad52 protein restores hRad51 binding to DNA. In the absence of any nucleotide cofactor where intrinsic binding of hRad51 to ssDNA is higher, hRad52 brings about no improved binding. hRad51 binding to ssDNA is strongly influenced by the DNA sequence. The protein binding to repeat sequences is poor compared to that of mixed DNA sequence. Interestingly, presence of hRad52 restores the ability of hRad51 binding to such DNA targets as well. Moreover, all the cooperative effects of hRad52 on hRad51 binding are highly specific to the latter’s binding to ssDNA and not to dsDNA. These results help us to model important mechanistic steps of hRad51 presynapsis on ssDNA templates

Human Rad52 facilitates a three-stranded pairing that follows no strand exchange: a novel pairing function of the protein

Human Rad52 protein, by analogy with the genetics of yeast Rad52, is believed to mediate a pathway of homologous recombination even independent of Rad51. Current study is focused on unraveling the molecular properties of hRad52 that endow the protein such an ability. We show here that the hRad52 protein binds single-stranded DNA (ssDNA) as well as 3'- and 5'-tailed duplexes severalfold better than blunt-ended duplexes, altering the sensitivity of the bound DNA to the action of DNase I. Protein binding is sensitive to the length of the ssDNA:  targets as short as a 33mer poorly bind the protein, whereas that of a 61mer and above bind the protein stably well. Such stable ssDNA−hRad52 complexes are highly competent in mediating not only the annealing of two complementary strands but also three-stranded pairing. The latter involves homologous recognition of linear duplex DNA by the ssDNA−hRad52 complex. We show that the hRad52 protein facilitates homologous recognition between ssDNA and duplex−DNA through a process that involves unwinding or transient unpairing of the interacting duplex via a novel three-stranded intermediate that does not lead to strand exchange. The results enable us to visualize a novel role for hRad52 that may model its function in a pathway requiring no hRad51

Interaction of hRad51 and hRad52 with MCM complex: a cross-talk between recombination and replication proteins

Human Rad51 and Rad52 are implicated in DNA repair during replication. Here we show, by pull-down assays, that purified hRad51 and hRad52 interact with each other as well as with Mini chromosome maintenance (MCM) proteins in HeLa cell extracts. Furthermore, immunoprecipitation experiments corroborate the same where hRad51 and hRad52 proteins not only cross-talk with each other but also pull down MCM3 and MCM2/3 proteins, respectively. The interaction scoring assays, performed reciprocally, demonstrate the same specificity, based on which, we speculate that MCM complex exhibits strong propensity to get physically recruited to the sites where hRad51 and hRad52-mediated homologously aligned ends need to be replicationally repaired

DNA mediated disassembly of hRad51 and hRad52 proteins and recruitment of hRad51 to ssDNA by hRad52

Purified human Rad51 and Rad52 proteins exhibit multiple oligomeric states, in vitro. Single-stranded DNA (ssDNA) renders high molecular weight aggregates of both proteins into smaller and soluble forms that include even the monomers. Consequently, these proteins that have a propensity to interact with each other's higher order forms by themselves, start interacting with monomeric forms in the presence of ssDNA, presumably reflecting the steps of protein assembly on DNA. In the same conditions, DNA binding assays reveal hRad52-mediated recruitment of hRad51 on ssDNA. Put together, these studies hint at DNA-induced disassembly of higher-order forms of Rad51 and Rad52 proteins as steps that precede protein assembly during hRad51 presynapsis on DNA, in vitro

The two Plasmodium falciparum nucleosome assembly proteins play distinct roles in histone transport and chromatin assembly

The malarial parasite Plasmodium falciparum has two nucleosome assembly proteins, PfNapS and PfNapL (Chandra, B. R., Olivieri, A., Silvestrini, F., Alano, P., and Sharma, A. (2005) Mol. Biochem. Parasitol. 142, 237-247). We show that both PfNapS and PfNapL interact with histone oligomers but only PfNapS is able to deposit histones onto DNA. This property of PfNapS is divalent cation-dependent and ATP-independent. Deletion of the terminal subdomains of PfNapS abolishes its nucleosome assembly capabilities, but the truncated protein retains its ability to bind histones. Both PfNapS and PfNapL show binding to the linker histone H1 suggesting their probable role in extraction of H1 from chromatin fibers. Our data suggests distinct sites of interaction for H1 versus H3/H4 on PfNapS. We show that PfNapS and PfNapL are phosphorylated both in vivo and in vitro by casein kinase-II, and this modification is specifically inhibited by heparin. Circular dichroism, fluorescence spectroscopy, and chymotrypsin fingerprinting data together suggest that PfNapL may undergo very small and subtle structural changes upon phosphorylation. Specifically, phosphorylation of PfNapL increases its affinity 3-fold for core histones H3, H4, and for the linker histone H1. Finally, we demonstrate that PfNapS is able to extract histones from both phosphorylated and unphosphorylated PfNapL, potentially for histone deposition onto DNA. Based on these results, we suggest that the P. falciparum NapL is involved in the nucleocytoplasmic relay of histones, whereas PfNapS is likely to be an integral part of the chromatin assembly motors in the parasite nucleus