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From Proteopedia

3D structure of E. coli XerD recombinase

Introduction

XerD is 298 amino acid protein that belongs, with its related partner recombinase (XerC), to the λ integrase family of site specific recombinases. XerD was identified through sequence homology with XerC, and is encoded in the same operon as recJ and DsbC (Blakely et al., 1993; Lovett and Kolodner, 1991; Missiakas et al., 1994). The enzymes of the integrase family use a conserved tyrosine nucleophile to mediate sequential strand exchange. A characteristic of tyrosine recombinases is their conserved group of four amino acids forming the active site. It consists of two arginines, one histidine, and one tyrosine (RHRY) which catalyze the recombination reaction. The two conserved arginines and the tyrosine are required for DNA cleavage, histidine is required for DNA rejoining.

An eventual odd number of homologous recombination events between newly replicated circular replicons (or during replication) leads to the production of dimeric chromosomes that will not be segregated between daughter cell and mother cell during division. To avoid this, E. coli has a Xer type site specific recombination system that contains a recombination site “dif” and two related tyrosine recombinases: XerC and XerD. They convert the circular multimers, that can arise through homologous recombination, into monomers. XerC and XerD each bind to half of the binding site (dif), left and right respectively, in a cooperative manner. Each protein cleaves a specific DNA strand.

Site specific recombinases cleave the DNA by a nucleophilic attack of a phosphodiester bond, exchange the two strands of double-stranded DNA involved, and perform the reattachment of DNA strands. λ integrases exchange single strands pairs in an ordered and sequential manner. More precisely, the conserved tyrosine hydroxyl of the conserved pattern RHRY attacks the scissile phosphate of a specific strand of each recombination site, forming a 3’ phosphotyrosil link (recombinase-DNA complex) and a free 5’OH end. In the second step, a 5’ hydroxyl from the adjacent partner duplex attacks the phosphotyrosyl bond to form a Holliday junction intermediate in which only two DNA strands are recombined. The recombination reaction is completed with the exchange of the second pair of strands using the same mechanism (cleavage / relegation), 6-8 bp away from the first site of strand exchanges. (Hallet and Sherratt , 1997).

XerC catalyzes the first strand exchange, thereby forming the Holliday junction, which is optionally resolved by XerD to generate recombinant products (Colloms et al., 1996).

Structure

XerD is separated in two domains: N-terminal domain consists of residues 1 to 107 whereas C-terminal domain consists residues 108 to 298. The first domain is an α-helical domain composed of four α helices (αA, αB, αC and αB) arranged like two parallel helix hairpins. This domain is responsible for the specific DNA binding thanks to the αB and αD helices. The second domain is composed of many α helices but also three-stranded antiparallel β-sheet. The last residues (271 – 298) forms a turn which is followed by a long α-helix, containing the active site tyrosine to which the DNA attaches covalently during the recombination reaction. This arrangement is made to support a cis cleavage mechanism (S. Subramanyal and all, 1997).

Structure and function

DNA interaction

XerD binding to its DNA target, involves major and minor groove interactions which wraps the DNA around XerD and induces a 40 degrees bend in the DNA. The DNA wraps around a helix-turn-helix motif which corresponds to helices: αG and αJ. These two helices are separated by 65 residues. XerD residues 220R and 221Q situated on αJ can specifically interact with DNA nucleotides on the dif site.

Domain 1 and domain 2 of XerD, both participate in DNA binding. Helices αB and αD of domain 1, interact with the major groove of the inner part of the recombinase binding site. In the domain 2, residues 236-245 interact with the minor groove on the “front” face of the DNA. The antiparallel strands β2 and β3 interact on the other side of the DNA. Conserved lysine residues at position 172 and 175 in the turn of the β2-β3 hairpin make the DNA backbone contacts on each of the DNA strands in the closeness of the scissile phosphate (S. Subramanyal and all, 1997).

Dif site (substrate on E.Coli)

The minimum size of the dif site for monomerization of dimeric chromosomes is 28 bp. It is composed of two 11-bp binding sites, where XerC and XerD bind, separated by a central region of 6 bp. Dif site requires no sequence or accessory proteins. The binding sites of the two recombinases are partial palindromes; however, the two half-sites are specifically recognized by XerC and XerD. The XerD binding sites are well preserved while XerC binding sites are quite variable. XerC binds more weakly to its respective site than XerD, which is compensated for by strong cooperative interactions between the two proteins.

Since six of the 11 positions are palindromic regarding the dif site, the binding determinants that are specific to either XerC or XerD are found in the five remaining positions. Nucleotides at position 10, 11, and 13 contributes to XerC-XerD binding discrimination.

The nucleotide at position +9 is the determinant factor for XerD binding. The presence of a T or G nucleotide determines the high affinity of the binding. The presence of a C nucleotide would lead to a weak interaction. The same goes for the nucleotide +13 which has to be A. (Hayes and Sherratt, 1997).

Mechanistic implications / interactions between XerC/XerD

The cleavage of a Holliday junction is performed by XerD via a cis mechanism. This reaction does not necessarily require the presence of XerC since the active site tyrosine Y279 is in close proximity to other residues of the active site.

However, efficient cleavage and strand exchange require the presence and the cooperative action of XerC and XerD. Even if the presence of the partner is not required for the nucleophilic activity, it is required to mediate the exchange of their strand. XerD binds to the right arm of the recombination site, resulting in a curvature in the DNA to assist in the binding of XerC to its respective sites (Blakeley and Sherratt, 1996). The C terminal helix, which contains the active tyrosine at one end, forms a major part of the interaction of XerD with XerC. XerD 263-267 residues are involved in the interaction with XerC and 256 – 258 residues are involved in XerC activation of catalysis.

Possible model of mechanism

Here is a possible simple mechanism to explain how the binding of the protein complex onto the recombination site causes the cleavage of DNA by the tyrosine active site. A change in the positioning of the alpha N, the C-terminal helix, induces a rotation of the Y279 side chain which shifts to a favorable position for the attack of the scissile phosphate. The binding of the associated recombinase can cause a conformational change that exposes the tyr279 inducing the active state of the protein (S. Subramanyal and all, 1997).

References

1. Blakely, G.W., May, G., McCulloch, R., Arciszewska, L., Burke, M., Lovett, S., and Sherratt, D.J. (1993).Two related recombinases are required for site specific recombination at dif and cer in Escherichia coli K12. Cell, 75, 351–361.

2. Blakely, G.W., and Sherratt, D.J. (1996). Cis and trans in site-specific recombination. Mol. Microbiol. 20, 233-238.

3. Colloms, S.D., McCulloch, R., Grant, K., Neilson, L., and Sherratt, D.J. (1996). Xer-mediated site-specific recombination in vitro. EMBO J. 15, 1172-1181.

4. Hallet, B., and Sherratt, D.J. (1997). Transposition and site-specific recombination: adapting DNA cut-and-paste mechanisms to a variety of genetic rearrangements. FEMS Microbiol. Rev., 21, 157-178.

5. Hayes, F., and Sherratt, D.J. (1997). Recombinase binding specificity at the chromosome dimer resolution site dif of Escherichia coli. J. Mol. Biol., 266, 525-537.

6. Lovett, S.T., and Kolodner, R.D. (1991). Nucleotide sequence of the Escherichia coli recJ chromosomal region and construction of RecJ overexpression plasmids. J. Bacteriol. 173, 363-364.

7. Missiakas, D., Georgopoulos, C. and Raina, S. (1994). The Escherichia coli dsbC (xprA) gene encodes a periplasmic protein involved in disulfide bond formation. EMBO J., 13, 2013-2020.

8. Hosahalli S.Subramanya, Lidia K.Arciszewska, Rachel A.Baker, Louise E.Bird, David J.Sherratt, and Dale B.Wigley (1997). Crystal structure of the site-specific recombinase, XerD.