Staying on message: ensuring fidelity in pre-mRNA splicing

The faithful expression of genes requires that cellular machinery select substrates with high specificity at each step in gene expression. High specificity is particularly important at the stage of nuclear pre-mRNA splicing, during which the spliceosome selects splice sites and excises intervening introns. With low specificity, the usage of alternative sites would yield insertions, deletions and frame shifts in mRNA. Recently, biochemical, genetic and genome-wide approaches have significantly advanced our understanding of splicing fidelity. In particular, we have learned that DExD/H-box ATPases play a general role in rejecting and discarding suboptimal substrates and that these factors serve as a paradigm for proofreading NTPases in other systems. Recent advances have also defined fundamental questions for future investigations

Replication-Dependent Unhooking of DNA Interstrand Cross-Links by the NEIL3 Glycosylase

During eukaryotic DNA interstrand cross-link (ICL) repair, cross-links are resolved (“unhooked”) by nucleolytic incisions surrounding the lesion. In vertebrates, ICL repair is triggered when replication forks collide with the lesion, leading to FANCI-FANCD2-dependent unhooking and formation of a double-strand break (DSB) intermediate. Using Xenopus egg extracts, we describe here a replication-coupled ICL repair pathway that does not require incisions or FANCI-FANCD2. Instead, the ICL is unhooked when one of the two N-glycosyl bonds forming the cross-link is cleaved by the DNA glycosylase NEIL3. Cleavage by NEIL3 is the primary unhooking mechanism for psoralen and abasic site ICLs. When N-glycosyl bond cleavage is prevented, unhooking occurs via FANCI-FANCD2-dependent incisions. In summary, we identify an incision-independent unhooking mechanism that avoids DSB formation and represents the preferred pathway of ICL repair in a vertebrate cell-free system

Ufd1-Npl4 recruit Cdc48 for disassembly of ubiquitylated CMG helicase at the end of chromosome replication

Disassembly of the Cdc45-MCM-GINS (CMG) DNA helicase is the key regulated step during DNA replication termination in eukaryotes, involving ubiquitylation of the Mcm7 helicase subunit, leading to a disassembly process that requires the Cdc48 “segregase”. Here, we employ a screen to identify partners of budding yeast Cdc48 that are important for disassembly of ubiquitylated CMG helicase at the end of chromosome replication. We demonstrate that the ubiquitin-binding Ufd1-Npl4 complex recruits Cdc48 to ubiquitylated CMG. Ubiquitylation of CMG in yeast cell extracts is dependent upon lysine 29 of Mcm7, which is the only detectable site of ubiquitylation both in vitro and in vivo (though in vivo other sites can be modified when K29 is mutated). Mutation of K29 abrogates in vitro recruitment of Ufd1-Npl4-Cdc48 to the CMG helicase, supporting a model whereby Ufd1-Npl4 recruits Cdc48 to ubiquitylated CMG at the end of chromosome replication, thereby driving the disassembly reaction

Replication-Dependent Unhooking of DNA Interstrand Cross-Links by the NEIL3 Glycosylase

During eukaryotic DNA interstrand cross-link (ICL) repair, cross-links are resolved (“unhooked”) by nucleolytic incisions surrounding the lesion. In vertebrates, ICL repair is triggered when replication forks collide with the lesion, leading to FANCI-FANCD2-dependent unhooking and formation of a double-strand break (DSB) intermediate. Using Xenopus egg extracts, we describe here a replication-coupled ICL repair pathway that does not require incisions or FANCI-FANCD2. Instead, the ICL is unhooked when one of the two N-glycosyl bonds forming the cross-link is cleaved by the DNA glycosylase NEIL3. Cleavage by NEIL3 is the primary unhooking mechanism for psoralen and abasic site ICLs. When N-glycosyl bond cleavage is prevented, unhooking occurs via FANCI-FANCD2-dependent incisions. In summary, we identify an incision-independent unhooking mechanism that avoids DSB formation and represents the preferred pathway of ICL repair in a vertebrate cell-free system

Eine prospektiv-randomisierte, placebokontrollierte Doppel-Blind-Studie und retrospektiver Vergleich zur Untersuchung des Einflusses oral verabreichter Lactobazillen und Bifidobakterien auf die drohende Frühgeburt

Die Frühgeburtlichkeit bedingt den Hauptanteil der intra-/ postnatalen Mortalität und frühen/späten Morbidität. Häufig in Folge einer Scheidenmilieustörung mit aufsteigender Genitalinfektion. Durch orale Zufuhr probiotischer Keime und Stabilisierung des Vaginalmilieus wäre ein positiver Therapieeffekt bei drohender Frühgeburt möglich. Sichtbar in Verlängerung der Tragzeit und Senkung der Frühgeburtenrate. 71 Schwangere erhielten neben dem Standardmanagement bis zur 37. SSW das Präparat/Placebo. Es zeigte sich für die Verumgruppe eine Tragzeitverlängerungstendenz ohne Signifikanz. Die Frühgeburtlichkeit war in beiden Gruppen gleich. Bei fast 40% der Frauen war die physiolog. Vaginalflora vermindert. Ein vaginaler Keimnachweis einhergehend mit pH-Erhöhung führte zum signifikanten Anstieg der Frühgeburtenrate und erhöhter Mortalität. Ein früher, evtl. prophylaktischer Einsatz von BB und LB scheint vielversprechend

Integrating DNA damage repair with the cell cycle

Abstract DNA is labile and constantly subject to damage. In addition to external mutagens, DNA is continuously damaged by the aqueous environment, cellular metabolites and is prone to strand breakage during replication. Cell duplication is orchestrated by the cell division cycle and specific DNA structures are processed differently depending on where in the cell cycle they are detected. This is often because a specific structure is physiological in one context, for example during DNA replication, while indicating a potentially pathological event in another, such as interphase or mitosis. Thus, contextualising the biochemical entity with respect to cell cycle progression provides information necessary to appropriately regulate DNA processing activities. We review the links between DNA repair and cell cycle context, drawing together recent advances

COVID-19, Equity and Men’s Health: Using Evidence to Inform Future Public Health Policy, Practice and Research Responses in Pandemics

In March 2020, the World Health Organization (WHO) announced that COVID-19 (novel coronavirus) reflected a global pandemic. Early epidemiological analyses demonstrated that boys and men have similar rates of COVID-19 infection to girls and women. However, boys and men appear to be disproportionately impacted with respect to severity and mortality, including those from marginalised or minority backgrounds. Yet, considerations of sex and gender, and their relationship to health and social inequities, have been absent from recent COVID-19 policy and practice pandemic responses. This evidence-based commentary discusses the nexus between COVID-19, equity, and boys and men’s health from a broad public health perspective. Using scholarship about intersections between race and gender; and poverty, social determinants of health, and gender; we explain why a health equity lens is important to address the health and social inequities boys and men face during pandemics. This contribution provides guidance about future global public healthpandemic responses for society’s most vulnerable groups of boys and men

The human DNA glycosylases NEIL1 and NEIL3 excise psoralen-induced DNA-DNA cross-links in a four-stranded DNA structure

Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specificities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks

A non‐proteolytic release mechanism for HMCES‐DNA‐protein crosslinks

The conserved protein HMCES crosslinks to abasic (AP) sites in ssDNA to prevent strand scission and the formation of toxic dsDNA breaks during replication. Here, we report a non‐proteolytic release mechanism for HMCES‐DNA‐protein crosslinks (DPCs), which is regulated by DNA context. In ssDNA and at ssDNA‐dsDNA junctions, HMCES‐DPCs are stable, which efficiently protects AP sites against spontaneous incisions or cleavage by APE1 endonuclease. In contrast, HMCES‐DPCs are released in dsDNA, allowing APE1 to initiate downstream repair. Mechanistically, we show that release is governed by two components. First, a conserved glutamate residue, within HMCES' active site, catalyses reversal of the crosslink. Second, affinity to the underlying DNA structure determines whether HMCES re‐crosslinks or dissociates. Our study reveals that the protective role of HMCES‐DPCs involves their controlled release upon bypass by replication forks, which restricts DPC formation to a necessary minimum