Tài liệu Báo cáo khoa học: Functional dissection of the Schizosaccharomyces pombe Holliday junction

Functional dissection of the Schizosaccharomyces pombe Holliday

junction resolvase Ydc2: in vivo role in mitochondrial DNA

maintenance

Barbara Sigala and Irina R. Tsaneva

Department of Biochemistry and Molecular Biology, University College London, London, UK

The crystal structure of the Schizosaccharomyces pombe

Holliday junction resolvase Ydc2 revealed significant struc￾tural homology with the Escherichia coli resolvase RuvC

but Ydc2 contains a small triple helical bundle that has

no equivalent in RuvC.Two of the a-helices that form this

bundle show homology to a putative DNA-binding motif

known as SAP.To investigate the biochemical function of

the triple-helix domain, truncated Ydc2 mutants were

expressed in E.coli and in fission yeast.Although the trun￾cated proteins retained all amino-acid residues that map to

the structural core of RuvC including the catalytic site,

deletion of the SAP motif alone or the whole triple-helix

domain of Ydc2 resulted in the complete loss of resolvase

activity and impaired significantly the binding of Ydc2 to

synthetic junctionsin vitro.These results are in full agreement

with our proposal for a DNA-binding role of the triple-helix

motif [Ceschini et al. (2001) EMBO J. 20, 6601–6611].The

biological effect of Ydc2 on mtDNA in yeast was probed

using wild-type and several Ydc2 mutants expressed in

Dydc2 S.pombe.The truncated mutants were shown to

localize exclusively to yeast mitochondria ruling out a

possible role of the helical bundle in mitochondrial targeting.

Cells that lacked Ydc2 showed a significant depletion of

mtDNA content.Plasmids expressing full-length Ydc2 but

not the truncated or catalytically inactive Ydc2 mutants

could rescue the mtDNA
phenotype.These results provide

evidence that the Holliday junction resolvase activity of

Ydc2 is required for mtDNA transmission and affects

mtDNA content in S.pombe.

Keywords: Holliday junction resolvase; mtDNA; yeast.

The Holliday junction is a key intermediate in homologous

recombination and double-strand break repair pathways

that proceeds via the reciprocal exchange of strands between

homologous DNA duplexes.Holliday junctions could also

arise from the regression of stalled replication forks [1–3]

and are thought to play an important role in the repair and

restart of stalled replication forks (reviewed in [1]).The

correct processing of this crossover intermediate is therefore

crucial for the integrity and maintenance of DNA in all

organisms including mitochondrial DNA (mtDNA).

Mitochondrial DNA amounts to about 15% of the DNA

content in Saccharomyces cerevisiae.A haploid cell contains

about 50 copies of the 75 kb mitochondrial genome as

clusters of linear concatamers (reviewed in [4]).Recombi￾nation between mtDNA genomes is common in S.cerevis￾iae and recombination intermediates were found to play an

important role for the faithful transmission of mtDNA in

this organism [5,6].Initially identified through mutations

that abolish the biased transmission of hypersuppressive

mtDNA [7], the CCE1 (MGT1) gene was shown to encode

a Holliday junction-resolving enzyme that functions exclu￾sively in mitochondria [8].The loss of this activity in

S.cerevisiae cce1 (mgt1) mutants resulted in the accumula￾tion of Holliday junctions in mtDNA [5] but the pathway

leading to the formation of the intermediates and their role

in mtDNA transmission is not fully understood.Homo￾logous recombination and replication are two of the most

fundamental processes in living cells and are tightly

interconnected [9,10].CCE1 could participate in a recom￾bination pathway associated with mtDNA replication, such

as intramolecular recombination (recombination with sister

chromatid) or in double-strand break repair.It could also

be involved in the initiation of recombination-dependent

replication events in mtDNA [4,11].CCE1 has a particular

effect on the partitioning of mtDNA and it has been

proposed that several mitochondrial genomes linked via

recombination junctions constitute the mtDNA heritable

unit in S.cerevisiae, whose size is affected directly by CCE1

[5].However, a recent study of the role of recombination in

mtDNA inheritance in S.cerevisiae showed that budding

cells were enriched in linear monomers of mtDNA.It was

proposed that in addition to a role in initiating a rolling￾circle DNA replication, recombination could be instrumen￾tal in resolving concatamers into linear monomers in the

process of mtDNA partitioning and transmission into buds

[11].

Several groups identified the Ydc2 protein of Schizosac￾charomyces pombe as a homologue of CCE1 [12–14].Like

CCE1 [7,15], Ydc2 (also called SpCCE1) localized exclu￾sively in mitochondria and did not affect nuclear DNA

Correspondence to I.Tsaneva, Department of Biochemistry,

University College London, Gower St., London WC1E 6BT, UK.

E-mail: [email protected]

Abbreviations: MtDNA, mitochondrial DNA; GFP, green fluorescent

protein; EGFP, enhanced green fluorescent protein; MTS, mito￾chondrial targeting signal.

(Received 21 February 2003, revised 28 April 2003,

accepted 12 May 2003)

Eur.J.Biochem. 270, 2837–2847 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03661.x