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RTF2 controls replication repriming and ribonucleotide excision at the replisome.

ABSTRACT: DNA replication through a challenging genomic landscape is coordinated by the replisome, which must adjust to local conditions to provide appropriate replication speed and respond to lesions that hinder its progression. We have previously shown that proteasome shuttle proteins, DNA Damage Inducible 1 and 2 (DDI1/2), regulate Replication Termination Factor 2 (RTF2) levels at stalled replisomes, allowing fork stabilization and restart. Here, we show that during unperturbed replication, RTF2 regulates replisome localization of RNase H2, a heterotrimeric enzyme that removes RNA from RNA-DNA heteroduplexes. RTF2, like RNase H2, is essential for mammalian development and maintains normal replication speed. However, persistent RTF2 and RNase H2 at stalled replication forks prevent efficient replication restart, which is dependent on PRIM1, the primase component of DNA polymerase α-primase. Our data show a fundamental need for RTF2-dependent regulation of replication-coupled ribonucleotide removal and reveal the existence of PRIM1-mediated direct replication restart in mammalian cells.

SUBMITTER: Conti BA

PROVIDER: S-EPMC10908796 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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RTF2 controls replication repriming and ribonucleotide excision at the replisome.

Conti Brooke A BA Ruiz Penelope D PD Broton Cayla C Blobel Nicolas J NJ Kottemann Molly C MC Sridhar Sunandini S Lach Francis P FP Wiley Tom F TF Sasi Nanda K NK Carroll Thomas T Smogorzewska Agata A

Nature communications 20240302 1

DNA replication through a challenging genomic landscape is coordinated by the replisome, which must adjust to local conditions to provide appropriate replication speed and respond to lesions that hinder its progression. We have previously shown that proteasome shuttle proteins, DNA Damage Inducible 1 and 2 (DDI1/2), regulate Replication Termination Factor 2 (RTF2) levels at stalled replisomes, allowing fork stabilization and restart. Here, we show that during unperturbed replication, RTF2 regula ...[more]

PMID: 38431617

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Project description:While hundreds of genes are induced by type I interferons, their roles in restricting the influenza virus life cycle remain mostly unknown. Using a loss-of-function CRISPR screen in cells prestimulated with interferon beta (IFN-β), we identified a small number of factors required for restricting influenza A virus replication. In addition to known components of the interferon signaling pathway, we found that replication termination factor 2 (RTF2) restricts influenza virus at the nuclear stage (and perhaps other stages) of the viral life cycle, based on several lines of evidence. First, a deficiency in RTF2 leads to higher levels of viral primary transcription, even in the presence of cycloheximide to block genome replication and secondary transcription. Second, cells that lack RTF2 have enhanced activity of a viral reporter that depends solely on four viral proteins that carry out replication and transcription in the nucleus. Third, when the RTF2 protein is mislocalized outside the nucleus, it is not able to restrict replication. Finally, the absence of RTF2 leads not only to enhanced viral transcription but also to reduced expression of antiviral factors in response to interferon. RTF2 thus inhibits primary influenza virus transcription, likely acts in the nucleus, and contributes to the upregulation of antiviral effectors in response to type I interferons.IMPORTANCE Viral infection triggers the secretion of type I interferons, which in turn induce the expression of hundreds of antiviral genes. However, the roles of these induced genes in controlling viral infections remain largely unknown, limiting our ability to develop host-based antiviral therapeutics against pathogenic viruses, such as influenza virus. Here, we performed a loss-of-function genetic CRISPR screen in cells prestimulated with type I interferon to identify antiviral genes that restrict influenza A virus replication. Besides finding key components of the interferon signaling pathway, we discovered a new restriction factor, RTF2, which acts in the nucleus, restricts influenza virus transcription, and contributes to the interferon-induced upregulation of known restriction factors. Our work contributes to the field of antiviral immunology by discovering and characterizing a novel restriction factor of influenza virus and may ultimately be useful for understanding how to control a virus that causes significant morbidity and mortality worldwide.

Dataset Information

RTF2 controls replication repriming and ribonucleotide excision at the replisome.

Publications

RTF2 controls replication repriming and ribonucleotide excision at the replisome.

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