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

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

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

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

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

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

TRAIP is a master regulator of DNA interstrand crosslink repair

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2–7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles

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

Author response

The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact modus operandi of Prp43 and of all other spliceosomal DEAH-box RNA helicases is still elusive. Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43ATP-analogRNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains. In the ATP-bound state this tunnel can be transformed into a groove prone for RNA binding by large rearrangements of the C-terminal domains. Several conformational changes between the ATP- and ADP-bound states explain the coupling of ATP hydrolysis to RNA translocation, mainly mediated by a ?-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases