{"id":124264,"date":"2021-06-25T16:23:25","date_gmt":"2021-06-25T23:23:25","guid":{"rendered":"https:\/\/lifeboat.com\/blog\/2021\/06\/pol%ce%b8-reverse-transcribes-rna-and-promotes-rna-templated-dna-repair"},"modified":"2021-06-25T16:23:25","modified_gmt":"2021-06-25T23:23:25","slug":"pol%ce%b8-reverse-transcribes-rna-and-promotes-rna-templated-dna-repair","status":"publish","type":"post","link":"https:\/\/lifeboat.com\/blog\/2021\/06\/pol%ce%b8-reverse-transcribes-rna-and-promotes-rna-templated-dna-repair","title":{"rendered":"Pol\u03b8 reverse transcribes RNA and promotes RNA-templated DNA repair"},"content":{"rendered":"

<\/a><\/p>\n

Genome-embedded ribonucleotides arrest replicative DNA polymerases (Pols) and cause DNA breaks. Whether mammalian DNA repair Pols efficiently use template ribonucleotides and promote RNA-templated DNA repair synthesis remains unknown. We find that human Pol\u03b8 reverse transcribes RNA, similar to retroviral reverse transcriptases (RTs). Pol\u03b8 exhibits a significantly higher velocity and fidelity of deoxyribonucleotide incorporation on RNA versus DNA. The 3.2-\u00c5 crystal structure of Pol\u03b8 on a DNA\/RNA primer-template with bound deoxyribonucleotide reveals that the enzyme undergoes a major structural transformation within the thumb subdomain to accommodate A-form DNA\/RNA and forms multiple hydrogen bonds with template ribose 2\u2032-hydroxyl groups like retroviral RTs. Last, we find that Pol\u03b8 promotes RNA-templated DNA repair in mammalian cells. These findings suggest that Pol\u03b8 was selected to accommodate template ribonucleotides during DNA repair.<\/p>\n

Polymerase \u03b8 (Pol\u03b8) is a unique DNA polymerase-helicase fusion protein in higher eukaryotes whose A-family polymerase domain evolved from Pol I enzymes (Fig. 1A<\/a>) (1<\/em><\/a>, 2<\/em><\/a>). However, contrary to most Pol I enzymes, Pol\u03b8 is highly error-prone and promiscuous (3<\/em><\/a>\u20136<\/em><\/a>), performs translesion synthesis (TLS) opposite DNA lesions (3<\/em><\/a>, 7<\/em><\/a>, 8<\/em><\/a>), and facilitates microhomology-mediated end-joining (MMEJ) of double-strand breaks (DSBs) by extending partially base-paired 3\u2032 single-stranded DNA (ssDNA) overhangs at DSB repair junctions (5<\/em><\/a>, 9<\/em><\/a>\u201312<\/em><\/a>). Pol\u03b8 is not expressed in most tissues but is highly expressed in many cancer cells, which corresponds to a poor clinical outcome (13<\/em><\/a>, 14<\/em><\/a>). Furthermore, Pol\u03b8 confers resistance to genotoxic cancer therapies and promotes the survival of cells deficient in DNA damage response pathways (11<\/em><\/a>, 13<\/em><\/a>\u201316<\/em><\/a>). Thus, Pol\u03b8 represents a promising cancer drug target.<\/p>\n

Intriguingly, Pol\u03b8 has an inactive proofreading domain due to acquired mutations (Fig. 1A<\/a>) (2<\/em><\/a>). Inactivating the 3\u2032-5\u2032 proofreading function of closely related A-family bacterial Pol I Klenow fragment (KF) enables this polymerase to reverse transcribe RNA like retroviral reverse transcriptases (RTs), which lack proofreading activity (fig. S1A) (17<\/em><\/a>, 18<\/em><\/a>). Because Pol\u03b8 is highly error-prone and promiscuous and contains an inactive proofreading domain, we hypothesized that it has RNA-dependent DNA synthesis activity. Given that ribonucleotides are the most frequently occurring nucleotide lesion in genomic DNA that arrest replicative Pols and cause DNA breaks (19<\/em><\/a>, 20<\/em><\/a>), we also envisaged that Pol\u03b8 would tolerate template ribonucleotides during its DNA repair activities and thus promote RNA-templated DNA repair synthesis (RNA-DNA repair). Although RNA-DNA repair mechanisms have been demonstrated in genetically engineered yeast cells (21<\/em><\/a>, 22<\/em><\/a>), they remain obscure in mammalian cells.<\/p>\n","protected":false},"excerpt":{"rendered":"

Genome-embedded ribonucleotides arrest replicative DNA polymerases (Pols) and cause DNA breaks. Whether mammalian DNA repair Pols efficiently use template ribonucleotides and promote RNA-templated DNA repair synthesis remains unknown. We find that human Pol\u03b8 reverse transcribes RNA, similar to retroviral reverse transcriptases (RTs). Pol\u03b8 exhibits a significantly higher velocity and fidelity of deoxyribonucleotide incorporation on RNA [\u2026]<\/p>\n","protected":false},"author":427,"featured_media":0,"comment_status":"open","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[11,412],"tags":[],"class_list":["post-124264","post","type-post","status-publish","format-standard","hentry","category-biotech-medical","category-genetics"],"_links":{"self":[{"href":"https:\/\/lifeboat.com\/blog\/wp-json\/wp\/v2\/posts\/124264","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/lifeboat.com\/blog\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/lifeboat.com\/blog\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/lifeboat.com\/blog\/wp-json\/wp\/v2\/users\/427"}],"replies":[{"embeddable":true,"href":"https:\/\/lifeboat.com\/blog\/wp-json\/wp\/v2\/comments?post=124264"}],"version-history":[{"count":0,"href":"https:\/\/lifeboat.com\/blog\/wp-json\/wp\/v2\/posts\/124264\/revisions"}],"wp:attachment":[{"href":"https:\/\/lifeboat.com\/blog\/wp-json\/wp\/v2\/media?parent=124264"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/lifeboat.com\/blog\/wp-json\/wp\/v2\/categories?post=124264"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/lifeboat.com\/blog\/wp-json\/wp\/v2\/tags?post=124264"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}