SUMMARY Bacterial group II intron reverse transcriptases (RTs) function in both intron mobility and RNA splicing and are evolutionary predecessors of retrotransposon, telomerase, and retroviral RTs, as well as spliceosomal protein Prp8 in eukaryotes. Here, we determined a crystal structure of a full-length thermostable group II intron RT in complex with an RNA template-DNA primer duplex and incoming dNTP at 3.0-Å resolution. We find that the binding of template-primer and key aspects of the RT active site are surprisingly different from retroviral RTs, but remarkably similar to viral RNA-dependent RNA polymerases. The structure reveals a host of features not seen previously in RTs that may contribute to distinctive biochemical properties of group II intron RTs, and it provides a prototype for many related bacterial and eukaryotic non-LTR-retroelement RTs. It also reveals how protein structural features used for reverse transcription evolved to promote the splicing of both group II and spliceosomal introns.

Graphical Abstract Highlights d E. coli carcinogen-like proteins cause DNA damage and mutation when upregulated d Human homologs form a cancer-predictive network, promote DNA damage and mutation d Conserved endogenous DNA damage-promoting mechanisms identified d DNA damage-up proteins (DDPs): a broad class of cancer gene function

Edited by Karin Musier-Forsyth This work was supported by National Institutes of Health Grants R01 GM37949 (to A. M. Lambowitz) and R35 GM131777 (to R. R.). Thermostable group II intron reverse transcriptase enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas and East Tennessee State University to InGex, LLC. A. M. Lambowitz, some former and present members of the Lambowitz laboratory, and the University of Texas are minority equity holders in InGex, LLC, and receive royalty payments from the sale of TGIRT enzymes and kits employing TGIRT template-switching activity for RNA-seq adapter addition and from the sublicensing of intellectual property to other companies. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article was selected as one of our Editors' Picks. This article contains Figs. S1-S10 and Tables S1-S3. RNA-Seq data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE138200.

Reverse transcriptases (RTs) can switch template strands during complementary DNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching (TS) plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-Seq, and may contribute to retroelement fitness by increasing evolutionary diversity and enabling continuous complementary DNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a TS complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template–DNA primer heteroduplex with a 1-nt 3′-DNA overhang. The structure showed that the 3′ end of the acceptor RNA binds in a pocket formed by an N-terminal extension present in non–long terminal repeat–retroelement RTs and the RT fingertips loop, with the 3′ nucleotide of the acceptor base paired to the 1-nt 3′-DNA overhang and its penultimate nucleotide base paired to the incoming dNTP at the RT active site. Analysis of structure-guided mutations identified amino acids that contribute to acceptor RNA binding and a phenylalanine residue near the RT active site that mediates nontemplated nucleotide addition. Mutation of the latter residue decreased multiple sequential template switches in RNA-Seq. Our results provide new insights into the mechanisms of TS and nontemplated nucleotide addition by RTs, suggest how these reactions could be improved for RNA-Seq, and reveal common structural features for TS by non–long terminal repeat–retroelement RTs and viral RNA–dependent RNA polymerases.

52 53 DNA damage provokes mutations and cancer, and results from external carcinogens or 54 endogenous cellular processes. Yet, the intrinsic instigators of DNA damage are poorly 55 understood. Here we identify proteins that promote endogenous DNA damage when 56 overproduced: the DNA-damaging proteins (DDPs). We discover a large network of DDPs 57 in Escherichia coli and deconvolute them into six DNA-damage-causing function clusters, 58 demonstrating DDP mechanisms in three: reactive-oxygen increase by transmembrane 59 transporters, chromosome loss by replisome binding, and replication stalling by transcription 60 factors. Their 284 human homologs are over-represented among known cancer drivers, and their 61 expression in tumors predicts heavy mutagenesis and poor prognosis. Half of tested human 62 homologs, when overproduced in human cells, promote DNA damage and mutation, with DNA-63 damaging mechanisms like those in E. coli. Together, our work reveals DDP networks that 64 provoke endogenous DNA damage and may indicate functions of many human known and newly 65 implicated cancer-promoting proteins. 66 67 68 69 DNA damage often underlies "spontaneous" mutations (Hastings et al.