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  • Review Article
  • Published:

The junction-resolving enzymes

Nature Reviews Molecular Cell Biology volume 2, pages 433–443 (2001)Cite this article

Key Points

  • Homologous recombination is an ancient process that functions to conserve genetic integrity and create genetic diversity. It relies on the formation of Holliday junctions — four-stranded DNA structures that create physical links between DNA duplexes. This review concentrates on the endonucleases that recognize this structure and catalyse its cleavage — the junction-resolving enzymes.

  • Recent progress includes the characterization of junction-resolving enzymes in the archaea and their viruses, the identification of the pox viral resolving enzyme responsible for resolution of viral concatamers, and the detection of a possible mammalian junction migrating and resolving complex analogous to the Escherichia coli RuvABC machinery

  • Studies in E. coli have emphasized the important role of homologous recombination in rescuing stalled or collapsed DNA replication forks, and the control of crossover versus non-crossover recombinant formation by the RuvABC complex. These findings are likely to be relevant in the eukaryotes.

  • Structural and sequence analysis indicates that most of the junction-resolving enzymes can be fitted into two main superfamilies of proteins. Sequence-selective enzymes including RuvC and CceI fall into the integrase class, whereas T7 endonuclease I and Hjc are classed with nucleases. All are probably evolved from a common metal-ion-binding domain.

  • Junction-resolving enzymes bind four-way DNA junctions in dimeric form. Binding is highly structure selective, but all the enzymes significantly distort the structure of the DNA on binding. Some of the resolving enzymes also have pronounced sequence selectivity at the cleavage stage; this can effectively ensure cleavage of Holliday junctions and not other branched DNA structures.

  • Several junction-resolving enzymes show an accelerated second strand cleavage, which is particularly pronounced for RuvC. This is probably the result of a strained DNA-enzyme complex, and ensures a productive resolution event.

  • The structures of four junction-resolving enzymes have been determined by crystallography. The protein folds have little in common. The active sites of T7 endonuclease I and Hjc are very similar to those of type II restriction enzymes, suggesting a two-metal-ion cleavage mechanism.

Abstract

Junction-resolving enzymes are ubiquitous nucleases that are important for DNA repair and recombination and act on DNA molecules containing branch points, especially four-way junctions. They show a pronounced selectivity for the structure of the DNA substrate but, despite its importance, the structural selectivity is not well understood. This poses an intriguing challenge in molecular recognition on a relatively large scale.

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Figure 1: Cleavage of DNA junctions by junction-resolving enzymes.
Figure 2: Homologous recombination and DNA replication.
Figure 3: Crystal structures of four junction-resolving enzymes.
Figure 4: Family tree of junction-resolving enzymes.
Figure 5: Proposed active site of endonuclease I.
Figure 6: Mechanism to account for the acceleration of second-strand cleavage.

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Authors and Affiliations

  1. Department of Biochemistry, CRC Nucleic Acid Structure Research Group, University of Dundee, Dundee, DD1 5EH , UK

    David M. J. Lilley

  2. Centre for Biomolecular Science, University of St Andrews , St Andrews, KY16 9ST, UK

    Malcolm F. White

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DATABASE LINKS

Rad51

Hjc

Cce1

RuvC

RusA

RecG

FURTHER INFORMATION

Lilley lab

White lab

ENCYCLOPEDIA OF LIFE SCIENCES

Eukaryotic recombination: initiation by double-strand breaks

Glossary

BRANCH MIGRATION

The process of exchange of base-pairing partners at a helical junction formed from homologous sequences.

INTEGRASE

The integrase protein of phage-λ is a tyrosine-recombinase protein that mediates the site-specific integration of the phage DNA into the host genome.

HETERODUPLEX

A DNA duplex formed by association between two homologous strands, each of which was previously hybridized to different complements. If the homology is less than 100%, the heteroduplex will contain base mismatches that will require repair.

GENETIC DRIFT

Random changes occurring in a gene over time, often with little or no phenotype.

RESTRICTION ENDONUCLEASES

Sequence-specific nucleases used by bacteria in defence against phage infection. These can be subclassified into different types according to their target and mode of action.

SUBUNIT-EXCHANGE RATES

In free solution, the subunits of junction-resolving enzymes may exchange between dimers. In general, this is characterized by a single exponential, giving the half-time for the exchange process. These vary widely for the known junction-resolving enzymes.

pI VALUE

The pH at which the ionization state of charged amino-acid side chains results in an overall electrical neutrality.

TRANSITION STATE

The point of highest energy along a reaction potential energy surface.

ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY

(EPR spectroscopy). Observation of the transitions between spin states of an unpaired electron in a magnetic field. The unpaired 3d5 electrons of manganese can be observed by EPR, providing information on the chemical environment of the metal ion.

ISOTHERMAL TITRATION CALORIMETRY

(ITC). Involves the measurement of heat absorbed or evolved as two interacting components are titrated in a cell. This provides a direct measure of the reaction enthalpy. The association constant can also be measured, from which other thermodynamic parameters can be calculated.

TN7 TRANSPOSASE

A polynucleotide transferase involved in the transposition of Tn7.

LEWIS ACID

A broader definition of acidity that encompasses any positively charged group, such as a metal cation.

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Lilley, D., White, M. The junction-resolving enzymes. Nat Rev Mol Cell Biol 2, 433–443 (2001). https://doi.org/10.1038/35073057x

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