Tài liệu Báo cáo Y học: Use of site-specific recombination as a probe of nucleoprotein complex

Use of site-specific recombination as a probe of nucleoprotein complex

formation in chromatin

Micha Schwikardi and Peter Dro¨ ge

Institute of Genetics, University of Cologne, Germany

DNA transactions in eukaryotes require that proteins gain

access to target sequences packaged in chromatin. Further,

interactions between distinct nucleoprotein complexes are

often required to generate higher-order structures. Here, we

employed two prokaryotic site-specific recombination sys￾tems to investigate how chromatin packaging affects the

assembly of nucleoprotein structures of different complex￾ities at more than 30 genomic loci. The dynamic nature of

chromatin permitted protein–DNA and DNA–DNA inter￾actions for sites of at least 34 bp in length. However, the

assembly of higher-order nucleoprotein structures on targets

spanning 114 bp was impaired. This impediment was

maintained over at least 72 h and was not affected by the

transcriptional status of chromatin nor by inhibitors of histone

deacetylases and topoisomerases. Our findings suggest that

nucleosomal linker-sized DNA segments become accessible

within hours for protein binding due to the dynamic nature

of chromatin. Longer segments, however, appear refractory

for complete occupancy by sequence-specific DNA-binding

proteins. The results thus also provide an explanation why

simple recombination systems such as Cre and Flp are

proficient in eukaryotic chromatin.

Keywords: chromatin; DNA reactivity; nucleoprotein com￾plex; site-specific recombination; transcription.

Alterations in chromatin structure are involved in the regu￾lation of DNA transactions such as transcription and site￾specific recombination. Recently, chromatin remodeling and

histone acetylation/deacetylation were identified as import￾ant regulators of chromatin structure at specific loci

(reviewed in [1–4]). Fundamental questions in this context

concern the general reactivity of DNA sites packaged into

chromatin. For example, does the assembly of complex

nucleoprotein structures require active chromatin remodel￾ing throughout the genome, or is remodeling only required

at specific loci? Further, the transcription process itself

transiently alters the structure of chromatin (reviewed in [5]).

Little is known, however, whether these dynamic alterations

render sequences in vivo more accessible for DNA-binding

proteins and, thus, contribute to the formation of complex

nucleoprotein structures.

Site-specific recombination has been used as a powerful

method to investigate fundamental questions both in pro￾karyotic and eukaryotic cells [6–10]. In our present study,

we sought to address the questions outlined above by

employing two site-specific recombination systems that differ

markedly in their complexity. The less elaborate system is

represented by the Cre recombinase encoded by Escherichia

coli phage P1. This enzyme is a member of the integrase

family of conservative site-specific recombinases and

functions efficiently in eukaryotic cells (reviewed in [11]).

Two Cre monomers bind cooperatively to a 34-bp recom￾bination sequence termed loxP (Fig. 1A). Collision of two

loxP-bound dimers results in the formation of a recombino￾genic complex that catalyzes two reciprocal single-strand￾transfer exchange reactions. This leads to deletion of

intervening DNA if two loxP sites are positioned as direct

repeats.

The second system employed in this study is derived from

the E. coli gd transposon-encoded resolvase. The resolvase

system is more complex than the Cre system. In the first step

leading to recombination resolvase binds to a recombination

sequence called res. A single res is composed of three

binding sites (I–III) for resolvase dimers which together

occupy 114 bp (Fig. 1B). Three dimers bind cooperatively

to res with comparable affinities towards sites I and II in

order to generate a recombinogenic complex, termed resolvo￾some [12]. Two resolvosomes then synapse by random

collision [13]. Two res must be present as direct repeats on

the same negatively supercoiled DNA molecule. Only this

site orientation leads to the formation of a functional

synaptic complex, termed synaptosome, which entraps three

(–)supercoils [14]. Strand exchange is catalyzed by dimers

bound at paired sites I, while those bound at sites II and III

serve accessory roles in synaptosome formation and in the

activation of strand cleavage therein (Fig. 1B). This rather

complex architecture imposes directionality on recombina￾tion, i.e. strand exchange always results in deletion of DNA

between two res.

Recently, we have transferred the gd system to higher

eukaryotes [10]. Two resolvases containing activating

mutations (E124Q or E102Y/E124Q) and a SV40-derived

nuclear localization signal (NLS) at their C-termini are

recombination-proficient on episomal DNA. Full res are still

Correspondence to P. Dro¨ge, Institute of Genetics, University of

Cologne, Weyertal 121, D-50931 Cologne, Germany.

Fax: 1 49 221470 5170, Tel.: 1 49 221470 3407,

E-mail: [email protected]

(Received 10 July 2001, revised 3 October 2001, accepted 5 October

2001)

Abbreviations: GFP, green fluorescence protein; Cre, cause of

recombination in phage P1; b-Gal, b-galactosidase; PGK, phospho

glycerate kinase; DMEM, Dulbecco’s modified Eagle’s medium; TRE,

tetracyclin-responsive-element; RLHRLZ, res-lox-hygromycin￾res-lox-lacZ.

Eur. J. Biochem. 268, 6256–6262 (2001) q FEBS 2001