Half a century of bacteriophage lambda recombinase: In vitro studies of lambda exonuclease and Red‐beta annealase

Abstract

DNA recombination, replication, and repair are intrinsically interconnected processes. From viruses to humans, they are ubiquitous and essential to all life on Earth. Single‐strand annealing homologous DNA recombination is a major mechanism for the repair of double‐stranded DNA breaks. An exonuclease and an annealase work in tandem, forming a complex known as a two‐component recombinase. Redβ annealase and λ‐exonuclease from phage lambda form the archetypal two‐component recombinase complex. In this short review article, we highlight some of the in vitro studies that have led to our current understanding of the lambda recombinase system. We synthesize insights from more than half a century of research, summarizing the state of our current understanding. From this foundation, we identify the gaps in our knowledge and cast an eye forward to consider what the next 50 years of research may uncover.

Structure of phage λ Redβ177 annealase shows how it anneals DNA strands during single-strand annealing homologous DNA recombination

“…The homo-oligomerization of Redβ has been reported by several groups using various approaches and techniques, including native mass spectrometry ( 27 ), electron microscopy ( 25, 59 ) and atomic force microscopy ( 11, 26 ). These results are briefly summarized above and in more detail in our recent review ( 16 ). Our reconstruction shows how Redβ 177 oligomerizes with substantial contacts between adjacent subunits in the helix, but there is limited top-bottom contacts between corresponding monomers in the next turn of the helix (Fig.…”

“…It has also emerged as a tool for genetic manipulation, termed recombineering ( 14, 15 ). There are currently two proposed models for how SSA may occur, which have been recently reviewed by us ( 16 ) and others ( 1, 17 ). In brief, variable processing of dsDNA ends by λExo leads to two different scenarios.…”

Viral Nucleases from Herpesviruses and Coronavirus in Recombination and Proofreading: Potential Targets for Antiviral Drug Discovery

“…Although some viruses with small circular genomes, such as the papovaviruses, have evolved to utilize host DNA replication machinery to replicate their genomes [ 17 ], larger DNA viruses have evolved to Recombination-Dependent Replication (RDR) during genome replication using virally encoded recombinases [ 5 ]. The best-characterized example of an exo/SSAP complex is phage λ Redα/β, which in addition to its role in viral replication, has been used to promote in vivo recombination-mediated genetic engineering or recombineering [ 2 , 18 , 19 ]. Interestingly, herpes simplex virus (HSV) also encodes an exo/SSAP comprised of the 5′-to-3′ alkaline exonuclease (UL12) and an ssDNA annealing protein (ICP8), and our laboratory has shown that both activities are essential for HSV DNA replication and the formation of replication compartments [ 4 , 6 , 20 , 21 ].…”

“…Extensive research on the mechanisms of recombination in both prokaryotic and eukaryotic cellular organisms has revealed a complex set of pathways by which genetic information is exchanged and repaired. Given the importance of these processes, it is perhaps not surprising that viruses have also evolved to rely heavily on recombination for genome replication and repair and to promote viral diversity [ 1 , 2 , 3 , 4 , 5 , 6 ]. Although most DNA and RNA viruses utilize recombination during replication, in this paper, we focus on Herpes and Coronaviruses, as these two viruses, in particular, have evolved efficient recombination pathways that specifically utilize virally encoded exonucleases.…”

DNA nanomachine for visual detection of structured RNA and double stranded DNA

“…Alternatively, λ exonuclease treatment is used to convert dsDNA to a more accessible ssDNA analyte. 11 Avoiding these stages is desirable both for fast, low temperature, and low-cost POC diagnostics as well as for the analysis of RNA under native conditions, e.g. , RNA monitoring in live cells.…”