The FLP recombinase of the Saccharomyces cerevisiae 2 microns plasmid attaches covalently to DNA via a phosphotyrosyl linkage.

Gronostajski, Richard M.; Sadowski, Paul D.

doi:10.1128/mcb.5.11.3274

Cited by 99 publications

(52 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The spacer has no direct FLP protein contacts within the central 6 bp (3, 25). The DNA is cleaved at the spacer boundaries, with FLP forming a 3'-phosphotyrosine intermediate (8). Sequence alterations in the spacer that maintain these sequence features are generally well tolerated by the system, with some constraints (26).…”

mentioning

confidence: 99%

Characterization of Holliday structures in FLP protein-promoted site-specific recombination.

Meyer-Leon¹,

Inman²,

Cox³

1990

Mol. Cell. Biol.

View full text Add to dashboard Cite

Holliday structures are formed in the course of FLP protein-promoted site-specific recombination. Here, we demonstrate that Holliday structures are formed in reactions involving wild-type substrates and that they are kinetically competent with respect to the overall reaction rate. Together with a previous demonstration of chemical competence (L. Meyer-Leon, L.-C. Huang, S. W. Umlauf, M. M. Cox, and R. B. Inman, Mol. Cefl. Biol. 8:3784-3796, 1988), Holliday structures therefore meet all criteria necessary to establish that they are obligate reaction intermediates in FLP-mediated site-specific recombination. In addition, kinetic evidence suggests that two distinct forms of the Holliday intermediate are present in the reaction pathway, interconverted in an isomerization process that is rate limiting at 0°C.The FLP protein is a site-specific recombinase encoded by the 2pLm plasmid of Saccharomyces cerevisiae (5). It belongs to the integrase family of site-specific recombinases, a family of eight known enzymes that includes the Cre protein of bacteriophage P1 and the Int protein of bacteriophage lambda (1,6,21,22). These recombinases share a limited degree of homology at the amino acid level and some similarities in the architecture of their respective recognition sites.The normal site of action (FLP recombination target [FRT]) of the FLP protein is a 48-base-pair (bp) DNA sequence that includes three 13-bp repeats arranged as shown in Fig. 1A, with an 8-bp spacer separating the first and second repeats. All three repeats are binding sites for FLP protein, although the third repeat can be deleted with no effect on in vitro recombination activity. The spacer has no direct FLP protein contacts within the central 6 bp (3, 25). The DNA is cleaved at the spacer boundaries, with FLP forming a 3'-phosphotyrosine intermediate (8). Sequence alterations in the spacer that maintain these sequence features are generally well tolerated by the system, with some constraints (26).Two reacting FRT sites must have homologous sequences in the spacer. This permits productive alignment of nonpalindromic FRT sites from only one orientation (24). FRT sites with symmetrical spacers may align in either of the two possible orientations with respect to the flanking DNA and form recombination products. One substrate with a symmetrical spacer, pJFS39, reacts approximately as efficiently as its wild-type counterpart, pJFS36 (Fig. 1A) (24).The FLP protein is stable and exhibits a slow catalytic turnover when glycerol and bovine serum albumin are added to the reaction mixture (7). Recombination also is observed when buffer and salt are the only solution additions besides DNA and protein (19) recombination sites (9,11,13,14,18,20). (Holliday structures are defined as four DNA strands that are derived from two duplex DNAs and connected by a covalently closed joint [10]). All of these recent studies utilized either substrates bearing nicks or mutations in the recognition site or recombinases with amino acid substitutions. For instance, wild-type FL...

show abstract

mentioning

confidence: 99%

Characterization of Holliday structures in FLP protein-promoted site-specific recombination.

Meyer-Leon¹,

Inman²,

Cox³

1990

Mol. Cell. Biol.

View full text Add to dashboard Cite

show abstract

“…It was observed that such plasmids always had segregated plasmid clones having the Sal/SalI and +/+ arrangements on the inverted VOL. 8,1988 on May 11, 2018 by guest http://mcb.asm.org/ Downloaded from repeats with significantly high frequencies, except for pSRT327 (R-), pSRT0609, and pSRT0627, each of which had a defect in the intramolecular recombination by disruption of the R gene or a deletion at the 58-bp region (Table 1). (Chimeric plasmid pSRT0627 has one exceptional clone exhibiting gene conversion, but it may be due to the gene conversion mechanism of the host cell).…”

Section: Resultsmentioning

confidence: 99%

Gene conversion associated with site-specific recombination in yeast plasmid pSR1.

Matsuzaki¹,

Araki²,

Oshima³

1988

Mol. Cell. Biol.

View full text Add to dashboard Cite

A circular DNA plasmid, pSRl, isolated from Zygosaccharomyces rouxii has a pair of inverted repeats consisting of completely homologous 959-base pair (bp) sequences. Intramolecular recombination occurs frequently at the inverted repeats in cells of Saccharomyces cerevisiae, as well as in Z. rouxii, and is catalyzed by a protein encoded by the R gene of its own genome. The recombination is, however, independent of the RAD52 gene of the host genome. A site for initiation of the intramolecular recombination in the S. cerevisiae host was delimited into, at most, a 58-bp region in the inverted repeats by using mutant plasmids created by linker insertion. The 58-bp region contains a pair with 14-bp dyad symmetry separated by a 3-bp spacer sequence. The recombination initiated at this site was accompanied by a high frequency of gene conversion (3 to 50% of the plasmid clones examined). Heterogeneity created by the linker insertion or by a deletion (at most 153 bp so far tested) at any place on the inverted repeats was converted to a homologous combination by the gene conversion, even in the rad52-1 mutant host. A mechanism implying branch migration coupled with DNA replication is discussed.

show abstract

“…8), indicating that the enzyme exhibits mechanistic similarities to integrase and Flp recombinase. These enzymes belonging to the integrase family of site-specific recombinases (36) bind covalently to the 3Ј-phosphoryl of the cleaved strand via a phosphotyrosyl linkage (37,38). Three short motifs named box A, B, and C conserved in these proteins (36) are also present in TelN.…”

Section: Discussionmentioning

confidence: 99%

Phage N15 Telomere Resolution

Deneke

Ziegelin

Lurz

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

The Escherichia coli prophage N15 exists as a linear DNA molecule with covalently closed ends. Purified N15 protelomerase TelN is the only protein required to convert circular DNA substrates to the linear form with hairpin termini. Within the center of the telomerase occupancy site tos, the target for TelN is the 56-bp telRL consisting of the central 22-bp palindrome telO and two 14-bp flanking inverted sequence repetitions. DNase I footprinting of TelN-telRL complexes shows a segment of ϳ50 bp protected by TelN. Surface plasmon resonance studies demonstrate that this extended footprint is caused by two TelN molecules bound to telRL. Stable TelN-target DNA complexes are achieved with telRL; however, the additional sequences of tos stabilize the TelN-target complexes. TelO alone is not sufficient for specific stable complex formation. However, processing can occur, i.e. generation of the linear covalently closed DNA. Within the context of telRL, sequences of telO are involved in specific TelN-telRL complex formation, in processing itself, and/or in recognition of the processing site. The sequence of the central (CG) 3 within telO that is part of a 14-bp stretch proposed to have Z-DNA conformation is essential for processing but not for formation of specific TelN-telRL complexes. The concerted action of both TelN molecules at the target site is the basis for telomere resolution. Capturing of reaction intermediates demonstrates that TelN binds covalently to the 3-phosphoryl of the cleaved strands.N15 is a lambdoid, temperate Escherichia coli phage that has double-stranded DNA with cohesive ends packaged in the virion (1). The complete nucleotide sequence of the 46-kb genome is known (2). Upon infection of E. coli, the DNA circularizes via the cos ends. In contrast to other temperate phages, N15 does not integrate into the host genome. Lysogeny is established by processing the telomere resolution site telRL to form a linear genome with covalently closed, hairpin-like ends telL and telR (see "Discussion"). The proposed telomerase occupancy site tos (1) contains a series of inverted repeats centered on the 56-bp palindromic telRL. This palindrome or part of it could form a potential cruciform structure that may function as the real substrate of the telomerase TelN. In vivo replication of linear N15 plasmids requires three N15-derived components as follows: the origin of replication ori, the replication initiation protein RepA, and TelN protein for resolution of the replicated telomeres (3). Ori probably is located immediately upstream of repA. repA is essential in N15 lytic replication and for maintenance of the linear prophage. Both lytic and lysogenic replication is independent of at least dnaA, dnaJ, dnaK, grpE, and recA (1, 4, 5). For telomere resolution, in vitro purified TelN and a telRL substrate are sufficient (6). In contrast to eukaryotic telomerases, TelN seems to be purely proteinaceous, lacking DNA synthesizing activity. The cleavingjoining activity of TelN resembles a specialized type of integrase that u...

show abstract

The FLP recombinase of the Saccharomyces cerevisiae 2 microns plasmid attaches covalently to DNA via a phosphotyrosyl linkage.

Cited by 99 publications

References 29 publications

Characterization of Holliday structures in FLP protein-promoted site-specific recombination.

Characterization of Holliday structures in FLP protein-promoted site-specific recombination.

Gene conversion associated with site-specific recombination in yeast plasmid pSR1.

Phage N15 Telomere Resolution

Contact Info

Product

Resources

About