Interactions of the Human MCM-BP Protein with MCM Complex Components and Dbf4

MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK

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Not AvailableIn the context of climate change, heat stress during the reproductive stages of chickpea (Cicer arietinum L.) leads to significant yield losses. In order to identify the genomic regions responsible for heat stress tolerance, a recombinant inbred line population derived from DCP 92-3 (heat sensitive) and ICCV 92944 (heat tolerant) was genotyped using the genotyping-by-sequencing approach and evaluated for two consecutive years (2017 and 2018) under normal and late sown or heat stress environments. A high-density genetic map comprising 788 single-nucleotide polymorphism markers spanning 1,125 cM was constructed. Using composite interval mapping, a total of 77 QTLs (37 major and 40 minor) were identified for 12 of 13 traits. A genomic region on CaLG07 harbors quantitative trait loci (QTLs) explaining >30% phenotypic variation for days to pod initiation, 100 seed weight, and for nitrogen balance index explaining >10% PVE. In addition, we also reported for the first time major QTLs for proxy traits (physiological traits such as chlorophyll content, nitrogen balance index, normalized difference vegetative index, and cell membrane stability). Furthermore, 32 candidate genes in the QTL regions that encode the heat shock protein genes, heat shock transcription factors, are involved in flowering time regulation as well as pollen-specific genes. The major QTLs reported in this study, after validation, may be useful in molecular breeding for developing heat-tolerant superior lines or varieties.Not Availabl