Biology subjects

Lentzsch, A. M.

Publications and source records attributed to Lentzsch, A. M..

3 recordsLinked to original sources

bioRxiv2021

Structural basis for template switching by a group II intron-encoded non-LTR-retroelement reverse transcriptase

Reverse transcriptases (RTs) can template switch during cDNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-seq, and may contribute to retroelement fitness by enabling continuous cDNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a template-switching complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template/DNA heteroduplex with a 1-nt 3-DNA overhang. The latter mimics a completed cDNA after non-templated addition (NTA) of a nucleotide complementary to the 3 nucleotide of the acceptor as required for efficient template switching. The structure showed that the 3 end of the acceptor RNA binds in a pocket formed by an N-terminal extension (NTE) present in non-long-terminal-repeat (LTR)-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 near the RT active site that mediates NTA. Mutation of the latter residue decreased multiple sequential template switches in RNA-seq. Our results provide new insights into the mechanisms of template switching and NTA by RTs, suggest how these reactions could be improved for RNA-seq, and reveal common structural features for template switching by non-LTR-retroelement RTs and viral RNA-dependent RNA polymerases.

biochemistry↗

bioRxiv2019

Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

The reverse transcriptases (RTs) encoded by mobile group II intron and other non-LTR-retro-elements differ from retroviral RTs in being able to template switch from the 5 end of one template to the 3 end of another without pre-existing complementarity between the donor and acceptor nucleic acids. Here, we used the ability of a thermostable group II intron RT (TGIRT; GsI-IIC RT) to template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3-DNA overhang end to establish a complete kinetic framework for the reaction and identify conditions that more efficiently capture acceptor RNAs or DNAs. The rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3-DNA overhang complementary to the 3 nucleotide of the acceptor RNA, suggesting a role for non-templated nucleotide addition of a complementary nucleotide to the 3 end of cDNAs synthesized from natural templates. Longer 3-DNA overhangs progressively decrease the rate of template switching, even when complementary to the 3 end of the acceptor template. The reliance on a single base pair with the 3 nucleotide of the acceptor together with discrimination against mismatches and the high processivity of the enzyme enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3 end. We discuss possible biological functions of the template-switching activity of group II intron and other non-LTR-retroelements RTs, as well as the optimization of this activity for adapter addition in RNA-and DNA-seq.

molecular biology↗

bioRxiv2018

Bacteria-to-human protein networks reveal origins of endogenous DNA damage

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

cancer biology↗