Functional complementation of UvsX and UvsY mutations in the mediation of T4 homologous recombination

Bacteriophage T4 homologous recombination events are promoted by presynaptic filaments of UvsX recombinase bound to single-stranded DNA (ssDNA). UvsY, the phage recombination mediator protein, promotes filament assembly in a concentration-dependent manner, stimulating UvsX at stoichiometric concentrations but inhibiting at higher concentrations. Recent work demonstrated that UvsX-H195Q/A mutants exhibit decreased ssDNA-binding affinity and altered enzymatic properties. Here, we show that unlike wild-type UvsX, the ssDNA-dependent ATPase activities of UvsX-H195Q/A are strongly inhibited by both low and high concentrations of UvsY protein. This inhibition is partially relieved by UvsY mutants with decreased ssDNA-binding affinity. The UvsX-H195Q mutant retains weak DNA strand exchange activity that is inhibited by wild-type UvsY, but stimulated by ssDNA-binding compromised UvsY mutants. These and other results support a mechanism in which the formation of competent presynaptic filaments requires a hand-off of ssDNA from UvsY to UvsX, with the efficiency of the hand-off controlled by the relative ssDNA-binding affinities of the two proteins. Other results suggest that UvsY acts as a nucleotide exchange factor for UvsX, enhancing filament stability by increasing the lifetime of the high-affinity, ATP-bound form of the enzyme. Our findings reveal new details of the UvsX/UvsY relationship in T4 recombination, which may have parallels in other recombinase/mediator systems

Performance and Carcass Traits of Market Beef Cattle Supplemented Self-Fed Byproducts on Pasture: Final Report

Over a two year period (2007 and 2008), 162 head of beef steers were finished with self-fed byproducts on cool season grass pastures. Yearling steers were continuously grazed at the Neely-Kinyon Farm in southwest Iowa on cool season grasses that were predominantly fescue at a stocking density of 2.25 head/acre. Half of the cattle were implanted (with Synovex®-S) or half were not. Cattle received a diet of either soyhulls-dried distillers grain with solubles (DDGS) or ground corn-dried distillers grains with solubles that was offered through self-feeders. The rations were mixed at a 1:1 ratio with a mineral balancer added which included Rumensin®. Live cattle performance and carcass traits were not affected by diet. Implanted cattle outgained non-implanted over the entire finishing period (3.52 lbs/d vs. 3.17 lbs/d). This led to implanted cattle coming off test heavier (1324 lbs vs. 1277 lbs) and railing with heavier carcasses (826 lbs vs. 800 lbs). Ribeye areas were greater (13.1 in2 vs. 12.7 in2) for implanted cattle; which was probably due to the heavier carcass weights. Non-implanted cattle had superior quality grades (55% vs. 40%) of low choice or better. Fatty acid profiles from the first year were analyzed and showed that raw beef samples from cattle on the soyhulls diet had significantly higher C18:2 In conclusion, pasture rearing cattle, when given access to self-fed by-products, provides for excellent performance on both live performance and carcass traits. Some considerations should be made by the feeder in regards to time of year when marketing cattle and the cattle’s genetics. This system is an alternative to high-grain conventional beef finishing production in feedlots

Performance and Carcass Traits of Market Beef Cattle Supplemented Self-Fed Byproducts on Pasture: A Progress Report

Over a two year period (2007 and 2008), 162 head of beef steers were finished with self-fed byproducts on cool season grass pastures. Yearling steers were continuously grazed at the Neely-Kinyon Farm in southwest Iowa on cool season grasses that were predominantly fescue at a stocking density of 2.25 head/acre. Half of the cattle were implanted (with Synovex®-S) or and half were not. Cattle received a diet of either soyhulls-dried distillers grain with solubles or corn-dried distillers grains with solubles (DDGS) that was offered as a meal through self-feeders. The rations were mixed in at 1:1 with a mineral balancer that included Rumensin®. Live cattle performance and carcass traits were not affected by diet. Implanted cattle outgained non-implanted over the entire finishing period (3.52 lbs/d vs. 3.17 lbs/d). This led to implanted cattle coming off test heavier (1324 lbs vs. 1277 lbs) and railing with heavier carcasses (826 lbs vs. 800 lbs). Ribeye areas were greater (13.1 in 2 vs. 12.7 in 2 ) for implanted cattle; which was probably due to the heavier carcass weights. Non-implanted cattle had superior quality grades (55% vs. 40%) of low choice or better. Year differences in quality grade (1023 vs. 985 in 2007 and 2008, respectively) were observed. This difference was attributed to factors that include genetic makeup of cattle, initial weights of cattle, time of year when cattle were harvested and grading technology. In conclusion, pasture rearing cattle, when given access to self-fed by-products, provides for excellent performance on both live performance and carcass traits. Some considerations should be made by the feeder in regards to time of year when marketing cattle and the cattle’s genetics. This system is an alternative to high-grain conventional beef finishing production in feedlots

Finishing Beef Cattle on Grass with Self-Fed By-Products 2006 Results

There has been increasing interest by consumers in beef from cattle that are finished or fattened “on grass” rather than in a conventional feedlot. Also recently, Iowa has had a proliferation of plants that produce ethanol from corn. One by-product of this process is distillers dried grains with solubles (DDGS). The objective of this study was to feed beef cattle to market weight by grazing cool-season grass supplemented with self-fed by-product pellets

Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

Homologous recombination (HR), a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR) processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR). T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms

Genome instability and pressure on non-homologous end joining drives chemotherapy resistance via a DNA repair crisis switch in triple negative breast cancer.

Chemotherapy is used as a standard-of-care against cancers that display high levels of inherent genome instability. Chemotherapy induces DNA damage and intensifies pressure on the DNA repair pathways that can lead to deregulation. There is an urgent clinical need to be able to track the emergence of DNA repair driven chemotherapy resistance and tailor patient staging appropriately. There have been numerous studies into chemoresistance but to date no study has elucidated in detail the roles of the key DNA repair components in resistance associated with the frontline clinical combination of anthracyclines and taxanes together. In this study, we hypothesized that the emergence of chemotherapy resistance in triple negative breast cancer was driven by changes in functional signaling in the DNA repair pathways. We identified that consistent pressure on the non-homologous end joining pathway in the presence of genome instability causes failure of the key kinase DNA-PK, loss of p53 and compensation by p73. In-turn a switch to reliance on the homologous recombination pathway and RAD51 recombinase occurred to repair residual double strand DNA breaks. Further we demonstrate that RAD51 is an actionable target for resensitization to chemotherapy in resistant cells with a matched gene expression profile of resistance highlighted by homologous recombination in clinical samples

The Tumor-Associated Variant RAD51 G151D Induces a Hyper-Recombination Phenotype

<div><p>The RAD51 protein plays a key role in the homology-directed repair of DNA double-strand breaks and is important for maintaining genome stability. Here we report on a novel human RAD51 variant found in an aggressive and therapy-refractive breast carcinoma. Expression of the RAD51 G151D variant in human breast epithelial cells increases the levels of homology-directed repair. Expression of RAD51 G151D in cells also promotes high levels of chromosomal aberrations and sister chromatid exchanges. <i>In vitro</i>, the purified RAD51 G151D protein directly and significantly enhances DNA strand exchange activity in the presence of RPA. In concordance with this result, co-incubation of G151D with BRCA2 resulted in a much higher level of strand-exchange activity compared to WT RAD51. Strikingly, the RAD51 G151D variant confers resistance to multiple DNA damaging agents, including ionizing radiation, mitomycin C, and doxorubicin. Our findings demonstrate that the RAD51 G151D somatic variant has a novel hyper-recombination phenotype and suggest that this property of the protein is important for the repair of DNA damage, leading to drug resistance.</p></div