Ribonucleotide Stress and Aging

National Institute On Aging

Investigators

Abstract

Mis-incorporated ribonucleotides are the most abundant type of genomic lesions, known to disrupt DNA transactions and cause replication stress. However, the mechanism(s) whereby these lesions affect molecular pathways of aging are yet to be examined. In a screen of replicative and DNA repair helicases, we found that RECQL1-catalyzed DNA unwinding was strongly inhibited by a single ribonucleotide in vitro, leaving RECQL1 trapped in the lesion vicinity. RECQL1 was enriched in the chromatin fraction of RNase H2 KO cells enriched for genomic mono-ribonucleotides, suggesting RECQL1 entrapment. Furthermore, DNA fiber and ƒ×-H2AX detection studies with human RNase H2-deficient cells suggest RECQL1 suppresses DNA damage by regulating fork speed when endogenous replication stress is elevated due to genomic ribonucleotides. To assess the impact of RECQL1: mono-ribonucleotide trapping on transcription, we will perform gene function enrichment analysis of RNA-sequencing reads to assess age-associated transcriptional profiles. To determine whether RECQL1 trapped by ribonucleotides is subject to proteolytic degradation in human cells, we will examine its chromatin fractionation following cellular treatment with proteasome inhibitors. These studies will assess if unchecked mono-ribonucleotide induced replication stress causes aberrant genomic changes associated with aging.