A novel multi-functional role for the MCM8/9 helicase complex in maintaining fork integrity during replication stress

The minichromosome maintenance (MCM) 8/9 helicase is a AAA+ complex involved in DNA replication-associated repair. Despite high sequence homology to the MCM2-7 helicase, an active role for MCM8/9 has remained elusive. We interrogated fork progression in cells lacking MCM8 or 9 and find there is a functional partitioning. Loss of MCM8 or 9 slows overall replication speed and increases markers of genomic damage and fork instability, further compounded upon treatment with hydroxyurea. MCM8/9 acts upstream and antagonizes the recruitment of BRCA1 in nontreated conditions. However, upon treatment with fork stalling agents, MCM9 recruits Rad51 to protect and remodel persistently stalled forks. The helicase function of MCM8/9 aids in normal replication fork progression, but upon excessive stalling, MCM8/9 directs additional stabilizers to protect forks from degradation. This evidence defines novel multifunctional roles for MCM8/9 in promoting normal replication fork progression and promoting genome integrity following stress.

Evaluation of the Genotoxicity in Fish Erythrocytes to Diagnose the Water Quality of Two Amazonian Estuaries Using the Micronucleus Test and Comet Assay

Genotoxicity studies in coastal ecosystems have been a priority in Environmental Risk Assessment (ERA). This research aimed to study the genotoxicity by the micronucleus test and comet assay in two Brazilian Amazon estuaries (anthropized and control) using Plagioscion squamosissimus as a bioindicator. Blood samples were collected from 54 specimens. No significant genotoxic effects were detected in the cells analyzed, although the highest occurrence was observed in anthropized site. The percentage of genomic damage differed between the sites studied, being always higher in anthropizes site as well. Of the nucleoids analyzed in this site, on average 28±14.42% of the cells were classified in the highest damage class (4). The fish analyzed in the present study are direct influence of xenobiont agents capable of producing damage to the genetic material of aquatic organisms in both sites and, consequently, may bring consequences still little reported in studies of morphophysiological alterations in humans.

Structure of a Ty1 restriction factor reveals the molecular basis of transposition copy number control

Excessive replication of Saccharomyces cerevisiae Ty1 retrotransposons is regulated by Copy Number Control, a process requiring the p22/p18 protein produced from a sub-genomic transcript initiated within Ty1 GAG. In retrotransposition, Gag performs the capsid functions required for replication and re-integration. To minimize genomic damage, p22/p18 interrupts virus-like particle function by interaction with Gag. Here, we present structural, biophysical and genetic analyses of p18m, a minimal fragment of Gag that restricts transposition. The 2.8 Å crystal structure of p18m reveals an all α-helical protein related to mammalian and insect ARC proteins. p18m retains the capacity to dimerise in solution and the crystal structures reveal two exclusive dimer interfaces. We probe our findings through biophysical analysis of interface mutants as well as Ty1 transposition and p18m restriction in vivo. Our data provide insight into Ty1 Gag structure and suggest how p22/p18 might function in restriction through a blocking-of-assembly mechanism.

High definition analyses of single cohort, whole genome sequencing data provides a direct route to defining sub-phenotypes and personalising medicine

ABSTRACTPossession of a clinical or molecular disease label alters the context in which life-course events operate, but rarely explains the phenotypic variability observed by clinicians. Whole genome sequencing of unselected endothelial vasculopathy patients demonstrated more than a third had rare, likely deleterious variants in clinically-relevant genes unrelated to their vasculopathy (1 in 10 within platelet genes; 1 in 8 within coagulation genes; and 1 in 4 within erythrocyte hemolytic genes). High erythrocyte membrane variant rates paralleled genomic damage and prevalence indices in the general population. In blinded analyses, patients with greater hemorrhagic severity that had been attributed solely to their vasculopathy had more deleterious variants in platelet (Spearman ρ=0.25, p=0.008) and coagulation (Spearman ρ=0.21, p=0.024) genes. We conclude that rare diseases can provide insights for medicine beyond their primary pathophysiology, and propose a framework based on rare variants to inform interpretative approaches to accelerate clinical impact from whole genome sequencing.

Evaluation of the genotoxicity in fish erythrocytes to diagnose the water quality of two Amazonian estuaries using the micronucleus test and comet assay

Genotoxicity studies in coastal ecosystems have been a priority in Environmental Risk Assessment (ERA). This research aimed to study the genotoxicity by the micronucleus test and comet assay in two Brazilian Amazon estuaries (anthropized and control) using Plagioscion squamosissimus as a bioindicator. Blood samples were collected from 54 specimens. No significant genotoxic effects were detected in the cells analyzed, although the highest occurrence was observed in anthropized site. The percentage of genomic damage differed between the sites studied, being always higher in anthropizes site as well. Of the nucleoids analyzed in this site, on average 28 ± 14.42% of the cells were classified in the highest damage class (4). The fish analyzed in the present study are direct influence of xenobiont agents capable of producing damage to the genetic material of aquatic organisms in both sites and, consequently, may bring consequences still little reported in studies of morphophysiological alterations in humans.

Recent Advances in Therapeutic Application of DNA Damage Response Inhibitors against Cancer

: DNA integrity is continuously challenged by intrinsic cellular processes and environmental agents. To overcome this genomic damage, cells have developed multiple signaling pathways collectively named as DNA damage response (DDR) and composed of three components: (i) sensor proteins, which detect DNA damage, (ii) mediators that relay the signal downstream and recruit the repair machinery, and (iii) the repair proteins, which restore the damaged DNA. A flawed DDR and failure to repair the damage lead to the accumulation of genetic lesions and increased genomic instability, which is recognized as a hallmark of cancer. Cancer cells tend to harbor increased mutations in DDR genes and often have fewer DDR pathways than normal cells. This makes cancer cells more dependent on particular DDR pathways and thus become more susceptible to compounds inhibiting those pathways compared to normal cells, which have all the DDR pathways intact. Understanding the roles of different DDR proteins in the DNA damage response and repair pathways and identification of their structures have paved the way for the development of their inhibitors as targeted cancer therapy. In this review, we describe the major participants of various DDR pathways, their significance in carcinogenesis, and focus on the inhibitors developed against several key DDR proteins.

Defining the Cell Wall, Cell Cycle and Chromatin Landmarks in the Responses of Brachypodium distachyon to Salinity

Excess salinity is a major stress that limits crop yields. Here, we used the model grass Brachypodium distachyon (Brachypodium) reference line Bd21 in order to define the key molecular events in the responses to salt during germination. Salt was applied either throughout the germination period (“salt stress”) or only after root emergence (“salt shock”). Germination was affected at ≥100 mM and root elongation at ≥75 mM NaCl. The expression of arabinogalactan proteins (AGPs), FLA1, FLA10, FLA11, AGP20 and AGP26, which regulate cell wall expansion (especially FLA11), were mostly induced by the “salt stress” but to a lesser extent by “salt shock”. Cytological assessment using two AGP epitopes, JIM8 and JIM13 indicated that “salt stress” increases the fluorescence signals in rhizodermal and exodermal cell wall. Cell division was suppressed at >75 mM NaCl. The cell cycle genes (CDKB1, CDKB2, CYCA3, CYCB1, WEE1) were induced by “salt stress” in a concentration-dependent manner but not CDKA, CYCA and CYCLIN-D4-1-RELATED. Under “salt shock”, the cell cycle genes were optimally expressed at 100 mM NaCl. These changes were consistent with the cell cycle arrest, possibly at the G1 phase. The salt-induced genomic damage was linked with the oxidative events via an increased glutathione accumulation. Histone acetylation and methylation and DNA methylation were visualized by immunofluorescence. Histone H4 acetylation at lysine 5 increased strongly whereas DNA methylation decreased with the application of salt. Taken together, we suggest that salt-induced oxidative stress causes genomic damage but that it also has epigenetic effects, which might modulate the cell cycle and AGP expression gene. Based on these landmarks, we aim to encourage functional genomics studies on the responses of Brachypodium to salt.