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Human replication protein A (RPA) is a heterotrimeric (70, 32, and 14 kDa subunits), eukaryotic single-stranded DNA (ssDNA) binding protein required for DNA recombination, repair, and replication. The three subunits of human RPA are composed of six conserved DNA binding domains (DBDs). Deletion and mutational studies have identified a high-affinity DNA binding core in the central region of the 70 kDa subunit, composed of DBDs A and B. To define the roles of each DBD in DNA binding, monomeric and tandem DBD A and B domain chimeras were created and characterized. Individually, DBDs A and B have a very low intrinsic affinity for ssDNA. In contrast, tandem DBDs (AA, AB, BA, and BB) bind ssDNA with moderate to high affinity. The AA chimera had a much higher affinity for ssDNA than did the other tandem DBDs, demonstrating that DBD A has a higher intrinsic affinity for ssDNA than DBD B. The RPA-DNA interface is similar in both DBD A and DBD B. Mutational analysis was carried out to probe the relative contributions of the two domains to DNA binding. Mutation of polar residues in either core DBD resulted in a significant decrease in the affinity of the RPA complex for ssDNA. RPA complexes with pairs of mutated polar residues had lower affinities than those with single mutations. The decrease in affinity observed when polar mutations were combined suggests that multiple polar interactions contribute to the affinity of the RPA core for DNA. These results indicate that RPA-ssDNA interactions are the result of binding of multiple nonequivalent domains. Our data are consistent with a sequential binding model for RPA, in which DBD A is responsible for positioning and initial binding of the RPA complex while DBD A together with DBD B direct stable, high-affinity binding to ssDNA.

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Agrawal DK. SOCS3 promotor hypermethylation and STAT3-NF-B interaction downregulate SOCS3 expression in human coronary artery smooth muscle cells. Am J Physiol Heart Circ Physiol 304: H776 -H785, 2013. First published January 18, 2013 doi:10.1152/ajpheart.00570.2012.-Suppressor of cytokine signaling-3 (SOCS3) is an intracellular negative regulator of cytokine signaling pathway. We recently found significant reduction in SOCS3 expression in coronary artery smooth muscle cells (CASMCs) of atherosclerotic swine and also in vitro cultured cells. Here, we investigated the underlying mechanisms of SOCS3 downregulation by IGF-1 and TNF-␣ in human CASMCs(hCASMCs). We propose that hypermethylation of CpG islands in the SOCS3 promoter is responsible for decrease in SOCS3 expression involving STAT3 and NFkB-p65 interaction. Western blot and qPCR data revealed significant upregulation of SOCS3 (6-to 10-fold) in hCASMC when treated individually with TNF-␣ (100 ng/ml) or IGF-1 (100 ng/ml). However, a significant decrease (5-fold) was observed by the combined treatment with TNF-␣ and IGF-1 compared with individual stimulation. IGF-1 phosphorylated STAT3 and TNF-␣-activated NF-B in hCASMCs. In the nuclear extract of hCASMCs stimulated with both TNF-␣ and IGF-1, there was an interaction between NF-B-p65 and pSTAT3, as determined by co-immunoprecipitation. Knockdown of STAT3 by small interfering RNA abolished SOCS3 expression in response to IGF-1. Methylation-specific PCR confirmed hypermethylation of SOCS3 promoter in hCASMCs stimulated with both TNF-␣ and IGF-1, and this was positively associated with elevated levels of DNA methyltransferase-I (9-to 10-fold). Knockdown of DNMT1 increased SOCS3 expression in IGF-1ϩTNF-␣-stimulated cells. Downregulation of SOCS3 in the presence of both TNF-␣ and IGF-1 in hCASMCs is due to SOCS3 promoter hypermethylation involving STAT3-NFkBp65 interaction. Because TNF-␣ and IGF-1 are released due to mechanical injury during coronary intervention, hypermethylation of SOCS3 gene could be an underlying mechanism of intimal hyperplasia and restenosis. cell signaling; cytokines; growth factors; hypermethylation; intimal hyperplasia; smooth muscle cells; suppressor of cytokine signaling 3; signal transducer and activator of transcription

Preparative-scale biotransformation of quercetin (1), fisetin (7), and (+)-catechin (12) with Streptomyces griseus (ATCC 13273) resulted in the isolation and characterization of nine known hydroxylated and/or methylated (2--6, 8, 9, 11, 13a) metabolites and two previously unknown (10 and 14) metabolites. S.griseus catalyzed aromatic hydroxylations of rings A and B of quercetin and fisetin. Mono- and dimethoxy ring-B metabolites were obtained with all three substrates. Methylation appeared to occur only when catechol functional groups were present. Metabolite structures were established by FABMS, EIMS, and 1D and 2D NMR analysis.