Double-strand DNA break repair and homologous recombination in Escherichia coli proceed by the RecBCD pathway, which is regulated by cis-acting elements known as sites. A crucial feature of this regulation is the RecBCD enzyme-directed loading of RecA protein specifically onto the 3-terminal, -containing DNA strand. Here we show that RecBC enzyme (lacking the RecD subunit) loads RecA protein constitutively onto the 3-terminal DNA strand, with no requirement for . This strand is preferentially utilized in homologous pairing reactions. We propose that RecA protein loading is a latent property of the RecBCD holoenzyme, which is normally blocked by the RecD subunit and is revealed following interaction with .

RhoA and its downstream target Rho kinase regulate serum response factor (SRF)-dependent skeletal and smooth muscle gene expression. We previously reported that long-term serum deprivation reduces transcription of smooth muscle contractile apparatus encoding genes, by redistributing SRF out of the nucleus. Because serum components stimulate RhoA activity, these observations suggest the hypothesis that the RhoA/Rho kinase pathway regulates SRF-dependent smooth muscle gene transcription in part by controlling SRF subcellular localization. Our present results support this hypothesis: cotransfection of cultured airway myocytes with a plasmid expressing constitutively active RhoAV14 selectively enhanced transcription from the SM22 and smooth muscle myosin heavy chain promoters and from a purely SRF-dependent promoter, but had no effect on transcription from the MSV-LTR promoter or from an AP2-dependent promoter. Conversely, inhibition of the RhoA/Rho kinase pathway by cotransfection with a plasmid expressing dominant negative RhoAN19, by cotransfection with a plasmid expressing Clostridial C3 toxin, or by incubation with the Rho kinase inhibitor, Y-27632, all selectively reduced SRF-dependent smooth muscle promoter activity. Furthermore, treatment with Y-27632 selectively reduced binding of SRF from nuclear extracts to its consensus DNA target, selectively reduced nuclear SRF protein content, and partially redistributed SRF from nucleus to cytoplasm, as revealed by quantitative immunocytochemistry. Treatment of cultured airway myocytes with latrunculin B, which reduces actin polymerization, also caused partial redistribution of SRF into the cytoplasm. Together, these results demonstrate for the first time that the RhoA/Rho kinase pathway controls smooth muscle gene transcription in differentiated smooth muscle cells, in part by regulating the subcellular localization of SRF. It is conceivable that the RhoA/Rho kinase pathway influences SRF localization through its effect on actin polymerization dynamics.

Homologous recombination and double-stranded DNA break repair in Escherichia coli are initiated by the multifunctional RecBCD enzyme. After binding to a double-stranded DNA end, the RecBCD enzyme unwinds and degrades the DNA processively. This processing is regulated by the recombination hot spot, Chi (: 5-GCT-GGTGG-3), which induces a switch in the polarity of DNA degradation and activates RecBCD enzyme to coordinate the loading of the DNA strand exchange protein, RecA, onto the single-stranded DNA products of unwinding. Recently, a single mutation in RecB, Asp-1080 3 Ala, was shown to create an enzyme (RecB D1080A CD) that is a processive helicase but not a nuclease. Here we show that the RecB D1080A CD enzyme is also unable to coordinate the loading of the RecA protein, regardless of whether sites are present in the DNA. However, the RecB D1080A CD enzyme does respond to sites by inactivating in a -dependent manner. These data define a locus of the RecBCD enzyme that is essential not only for nuclease function but also for the coordination of RecA protein loading.

Genetic recombination in Escherichia coil is stimulated by a RecBCD enzyme-mediated event at DNA sequences known as Chi (X) sites (5'-GCTGGTGG-3'). Previously, it was shown that X acts to regulate the nuclease activity of RecBCD; here, we demonstrate that, under appropriate conditions, interaction with X sites can also result in an inactivation of helicase activity of RecBCD (ssDNA) and double-stranded DNA (dsDNA) by means of its ATP-dependent exonuclease activities and its ATP-stimulated ssDNA endonuclease activity. In addition to these degradative activities, the enzyme is a DNA helicase that can processively unwind large tracts of dsDNA (>30 kb) at rates up to 1000 bp/sec (2-4). During dsDNA unwinding, the ATP-dependent degradation ofDNA is asymmetric, with the 3'-terminal DNA strand at the entry site for RecBCD being degraded more vigorously than the 5'-terminal strand (5-8).The X recombination hotspots are composed of the DNA sequence 5'-GCTGGTGG-3' (9, 10); recombination events are 5-10 times more frequent near X than far from it and these exchanges are recBC-dependent (11-13). Stimulation of recombination by X occurs primarily downstream (5' side) of the X site and extends, with decreasing magnitude, for >10 kb from X (12, 14). In vitro, recognition of X by RecBCD results in a single-strand break in the DNA strand containing the X sequence, 4-6 nucleotides to the 3' side of the X sequence; this occurs during the unwinding ofdsDNA and is orientation dependent (15, 16). These properties led to models which proposed the interaction between RecBCD and X served to initiate the RecA-dependent DNA strand invasion (17, 18).Recently, the formation of kspecific homologously paired joint molecules was demonstrated in vitro using a reconstituted recombination reaction consisting of purified RecA, RecBCD, and ssDNA-binding protein (SSB) (7). These results supported most predictions of the nick-initiation model (18), but they also uncovered an added feature of the RecBCD-X interaction (7,8). X recognition resulted in attenuation of the nuclease, but not the helicase, activity of RecBCD (7, 8 The biochemical basis for this x-dependent attenuation of nuclease activity remained unknown; however, the interpretations of genetic experiments propose a testable hypothesis (19,20). To explain the hyper-recombination phenotype of recD null mutations, it was proposed that interaction with a X site activates the nonrecombinogenic RecBCD by the removal of the RecD subunit, producing a RecBC(D-) protein that is changed to become a recombinogenic resolver of Holliday junctions (19,20). In this report, we describe experiments which test the RecD subunit-removal concept. We demonstrate that during the process of DNA unwinding at conditions of low nuclease activity (i.e., low Mg2+ concentration), the specific protein-DNA interaction between RecBCD and X causes a reversible inactivation of RecBCD's helicase activity and, we presume, its nuclease activity as well. This x-specific inhibition is fully reversible, and all RecBCD ac...

Background: Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme, and is stimulated by DNA elements known as Chi (x) sites. The RecBCD enzyme is both a helicase and a nuclease. Recognition of x causes both attenuation of the 3 0 →5 0 exonuclease activity of the RecBCD enzyme, and activation of an exonuclease activity with 5 0 →3 0 polarity, while leaving the helicase activity unaffected. A variety of evidence suggests that x-recognition by RecBCD enzyme is accompanied by ejection of the RecD subunit.