RNF212B E3 ligase is essential for crossover designation and maturation during male and female meiosis in the mouse.

Meiosis, a reductional cell division, relies on precise initiation, maturation, and resolution of crossovers (COs) during prophase I to ensure the accurate segregation of homologous chromosomes during metaphase I. This process is regulated by the interplay of RING-E3 ligases such as RNF212 and HEI10 in mammals. In this study, we functionally characterized a recently identified RING-E3 ligase, RNF212B. RNF212B colocalizes and interacts with RNF212, forming foci along chromosomes from zygonema onward in a synapsis-dependent and DSB-independent manner. These consolidate into larger foci at maturing COs, colocalizing with HEI10, CNTD1, and MLH1 by late pachynema. Genetically, RNF212B foci formation depends on Rnf212 but not on Msh4, Hei10, and Cntd1, while the unloading of RNF212B at the end of pachynema is dependent on Hei10 and Cntd1. Mice lacking RNF212B, or expressing an inactive RNF212B protein, exhibit modest synapsis defects, a reduction in the localization of pro-CO factors (MSH4, TEX11, RPA, MZIP2) and absence of late CO-intermediates (MLH1). This loss of most COs by diakinesis results in mostly univalent chromosomes. Double mutants for Rnf212b and Rnf212 exhibit an identical phenotype to that of Rnf212b single mutants, while double heterozygous demonstrate a dosage-dependent reduction in CO number, indicating a functional interplay between paralogs. SUMOylome analysis of testes from Rnf212b mutants and pull-down analysis of Sumo- and Ubiquitin-tagged HeLa cells, suggest that RNF212B is an E3-ligase with Ubiquitin activity, serving as a crucial factor for CO maturation. Thus, RNF212 and RNF212B play vital, yet overlapping roles, in ensuring CO homeostasis through their distinct E3 ligase activities.

Assembly and distributive action of an archaeal DNA polymerase holoenzyme.

The assembly and enzymatic ability of the replication DNA polymerase holoenzyme from Sulfolobus solfataricus (Sso) was investigated using presteady-state fluorescence resonance energy transfer assays coupled with functional and structural studies. Kinetic experiments reveal that ATP binding to replication factor C (RFC) is sufficient for loading the heterotrimeric PCNA123 [proliferating cell nuclear antigen (PCNA)] clamp onto DNA that includes a rate-limiting conformational rearrangement of the complex. ATP hydrolysis is required for favorable recruitment and interactions with the replication polymerase (PolB1) that most likely include clamp closing and RFC dissociation. Surprisingly, the assembled holoenzyme complex synthesizes DNA distributively and with low processivity, unlike most other well-characterized DNA polymerase holoenzyme complexes. We show that PolB1 repeatedly disengages from the DNA template, leaving PCNA123 behind. Interactions with a newly identified C-terminal PCNA-interacting peptide (PIP) motif on PolB1 specifically with PCNA2 are required for holoenzyme formation and continuous re-recruitment during synthesis. The extended tail-like structure of the C-terminal PIP motif in PolB1 is revealed alone and when bound to DNA using small-angle X-ray scattering allowing us to develop a model for the holoenzyme complex. This is the first detailed kinetic description of clamp loading and holoenzyme assembly in crenarchaea and has revealed a novel mode for dynamic processivity that occurs by a polymerase exchange mechanism. This work has important implications for processive DNA replication synthesis and also suggests a potential mechanism for polymerase switching to bypass lesions.