Deepa Subramanian

hMSH2–hMSH6 Forms a Hydrolysis-Independent Sliding Clamp on Mismatched DNA

Mismatch recognition by the human MutS homologs hMSH2-hMSH6 is regulated by adenosine nucleotide binding, supporting the hypothesis that it functions as a molecular switch. Here we show that ATP-induced release of hMSH2-hMSH6 from mismatched DNA is prevented if the ends are blocked or if the DNA is circular. We demonstrate that mismmatched DNA provokes ADP-->ATP exchange, resulting in a discernible conformational transition that converts hMSH2-hMSH6 into a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone. Our results support a model for bidirectional mismatch repair in which stochastic loading of multiple ATP-bound hMSH2-hMSH6 sliding clamps onto mismatch-containing DNA leads to activation of the repair machinery and/or other signaling effectors similar to G protein switches.

The Processing of Holliday Junctions by BLM and WRN Helicases Is Regulated by p53

BLM, WRN, and p53 are involved in the homologous DNA recombination pathway. The DNA structure-specific helicases, BLM and WRN, unwind Holliday junctions (HJ), an activity that could suppress inappropriate homologous recombination during DNA replication. Here, we show that purified, recombinant p53 binds to BLM and WRN helicases and attenuates their ability to unwind synthetic HJ in vitro. The p53 248W mutant reduces abilities of both to bind HJ and inhibit helicase activities, whereas the p53 273H mutant loses these abilities. Moreover, full-length p53 and a C-terminal polypeptide (residues 373–383) inhibit the BLM and WRN helicase activities, but phosphorylation at Ser376 or Ser378 completely abolishes this inhibition. Following blockage of DNA replication, Ser15 phospho-p53, BLM, and RAD51 colocalize in nuclear foci at sites likely to contain DNA replication intermediates in cells. Our results are consistent with a novel mechanism for p53-mediated regulation of DNA recombinational repair that involves p53 post-translational modifications and functional protein-protein interactions with BLM and WRN DNA helicases.

Mechanism of topology simplification by type II DNA topoisomerases

Type II DNA topoisomerases actively reduce the fractions of knotted and catenated circular DNA below thermodynamic equilibrium values. To explain this surprising finding, we designed a model in which topoisomerases introduce a sharp bend in DNA. Because the enzymes have a specific orientation relative to the bend, they act like Maxwells demon, providing unidirectional strand passage. Quantitative analysis of the model by computer simulations proved that it can explain much of the experimental data. The required sharp DNA bend was demonstrated by a greatly increased cyclization of short DNA fragments from topoisomerase binding and by direct visualization with electron microscopy.

p53 binds telomeric single strand overhangs and t-loop junctions in vitro

The interaction of p53 with a human model telomere in vitro was examined by electron microscopy. p53 demonstrated a sequence-independent affinity for telomeric DNA in vitro, localizing to the 3′ single strand overhang and the t-loop junction both in the presence and absence of associated TRF2. Binding was not observed above background along the duplex telomeric repeats. However, the efficiency of TRF2-catalyzed t-loop formation on the model DNA was increased 2-fold in the presence of p53 although a variety of single strand or Holliday junction-binding proteins did not facilitate t-loop formation. These results suggest that p53 has an active role in telomere maintenance and structure through association with the t-loop junction.

Analysis of the Binding of p53 to DNAs Containing Mismatched and Bulged Bases

The tumor suppressor protein p53 modulates cellular response to DNA damage by a variety of mechanisms that may include direct recognition of some forms of primary DNA damage. Linear 49-base pair duplex DNAs were constructed containing all possible single-base mismatches as well as a 3-cytosine bulge. Filter binding and gel retardation assays revealed that the affinity of p53 for a number of these lesions was equal to or greater than that of the human mismatch repair complex, hMSH2-hMSH6, under the same binding conditions. However, other mismatches including G/T, which is bound strongly by hMSH2-hMSH6, were poorly recognized by p53. The general order of affinity of p53 was greatest for a 3-cytosine bulge followed by A/G and C/C mismatches, then C/T and G/T mismatches, and finally all the other mismatches.

p53 Monitors Replication Fork Regression by Binding to “Chickenfoot” Intermediates

The tumor suppressor protein, p53, utilizes multiple mechanisms to ensure faithful transmission of the genome including regulation of DNA replication, repair, and recombination. Monitoring these pathways may involve direct binding of p53 to the DNA intermediates of these processes. In this study, we generated templates resembling stalled replication forks and utilized electron microscopy to examine p53 interactions with these substrates. Our results show that p53 bound with high affinity to the junction of stalled forks, whereas two cancer-derived p53 mutants showed weak binding. Additionally, some of the templates were rearranged to form “chickenfoot” structures in the presence of p53. These were mostly formed due to p53 trapping intermediates of spontaneous fork regression; however, in a small population, the protein appeared to be promoting their formation. Collectively, these results demonstrate the importance of sequence-independent binding in p53-mediated maintenance of genomic integrity.

Interplay between TAp73 Protein and Selected Activator Protein-1 (AP-1) Family Members Promotes AP-1 Target Gene Activation and Cellular Growth

Background: TAp73, which is overexpressed in cancers, activates AP-1 target genes. Results: TAp73 binds to c-Jun on the chromatin around TRE sites on AP-1 target promoters, leading to recruitment of other AP-1 family members. Conclusion: Interaction of TAp73 with selected AP-1 members enhances target gene activation and cellular growth. Significance: c-Jun-dependent cooperativity between TAp73 and selected AP-1 members contributes to cellular growth. Unlike p53, which is mutated at a high rate in human cancers, its homologue p73 is not mutated but is often overexpressed, suggesting a possible context-dependent role in growth promotion. Previously, we have shown that co-expression of TAp73 with the proto-oncogene c-Jun can augment cellular growth and potentiate transactivation of activator protein (AP)-1 target genes such as cyclin D1. Here, we provide further mechanistic insights into the cooperative activity between these two transcription factors. Our data show that TAp73-mediated AP-1 target gene transactivation relies on c-Jun dimerization and requires the canonical AP-1 sites on target gene promoters. Interestingly, only selected members of the Fos family of proteins such as c-Fos and Fra1 were found to cooperate with TAp73 in a c-Jun-dependent manner to transactivate AP-1 target promoters. Inducible expression of TAp73 led to the recruitment of these Fos family members to the AP-1 target promoters on which TAp73 was found to be bound near the AP-1 site. Consistent with the binding of TAp73 and AP-1 members on the target promoters in a c-Jun-dependent manner, TAp73 was observed to physically interact with c-Jun specifically at the chromatin via its carboxyl-terminal region. Furthermore, co-expression of c-Fos or Fra1 was able to cooperate with TAp73 in potentiating cellular growth, similarly to c-Jun. These data together suggest that TAp73 plays a vital role in activation of AP-1 target genes via direct binding to c-Jun at the target promoters, leading to enhanced loading of other AP-1 family members, thereby leading to cellular growth.