Early use of sildenafil after RRP may preserve intracorporeal SM content. At higher doses post-RRP sildenafil may increase SM content. The effect on the return of potency is not known but maintaining the pro-erectile ultrastructure is integral to rehabilitating post-RRP erectile function.

Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4–5 MCM hexamers, and 2 Cdc6. Relative to these subunits, ∼0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain ∼70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of existing Cdc45 during S-phase.

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SUMMARY Microphthalmia-associated transcription factor (MITF) is a survival factor in melanocytes and melanoma cells. MITF regulates expression of anti-apoptotic genes and promotes lineage specific survival in response to ultraviolet (UV) radiation and to chemotherapeutics. SWI/SNF chromatin remodeling enzymes interact with MITF to regulate MITF target gene expression. We determined that the catalytic subunit, BRG1, of the SWI/SNF complex protects melanoma cells against UV-induced death. BRG1 prevents apoptosis in UV- irradiated melanoma cells by activating expression of the melanoma inhibitor of apoptosis (ML-IAP). Down-regulation of ML-IAP compromises BRG1 mediated survival of melanoma cells in response to UV-radiation. BRG1 regulates ML-IAP expression by cooperating with MITF to promote transcriptionally permissive chromatin structure on the ML-IAP promoter. The alternative catalytic subunit, BRM and the BRG1 associated factor, BAF180 were found to be dispensable for elevated expression of ML-IAP in melanoma cells. Thus, we illuminate a lineage-specific mechanism by which a specific SWI/SNF subunit, BRG1, modulates the cellular response to DNA damage by regulating an anti-apoptotic gene and implicate this subunit of the SWI/SNF complex in mediating the pro-survival function of MITF. SIGNIFICANCE SWI/SNF enzymes interact with the Microphthalmia-associated transcription factor (MITF) a lineage addiction oncogene, to promote MITF target gene expression in melanoma cells. In this study we determined that the SWI/SNF component, BRG1, promotes melanoma survival in response to UV-radiation, by activating expression of the melanoma inhibitor of apoptosis, ML-IAP gene. Our data show that BRG1 and MITF co-operate to establish permissive chromatin structure on the ML-IAP promoter and alter the association of other epigenetic regulators. Thus, we have elucidated a mechanism by which a component of the SWI/SNF complex promotes the pro-survival function of MITF. We further demonstrate that the BRG1 associated factor, BAF180, is not required for the activation of ML-IAP, suggesting that a specific configuration of the SWI/SNF complex mediates distinct activities. These results provide insight into how SWI/SNF function is deregulated in melanoma.