Worm Breeder's Gazette 12(5): 22 (February 1, 1993)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Tc1 Transposase has Separate Domains for Site-Specific and General DNA Binding

J. Chris Vos, Henri G.A.M. van Luenen, Ronald H.A. Plasterk

The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX

We have previously reported the intron-exon structure of the

putative transposase gene Tc1A )of the transposable element Tc1

and the binding of Tc1A to the inverted repeat of Tc1 (WBG 12 (3),

pg. 87). In a manner similar to Tc3 (see WBG 12(4), pg. 15), we

analyzed somatic transposition of endogenous Tc1 elements in

transgenic N2 animals in which we induced Tc1 A expression. In non-

transgenic, or in non-induced transgenic N2 ,we found approximately

3 Tc1 insertions in the gpa-2 gene per 1 ug of worm DNA

(equivalent to approximately 10.000 genomes). A 15 fold increase in

the number of Tc1 insertions is seen after Tc1 A expression in the

transgenic strain. Therefore, we conclude that the Tc1A gene

product is a transposase and that it is a limiting factor for

transposition in N2 . Tc1 A induction does not result in Tc3

transposition, which requires expression of its own Tc3

transposase. We expressed Tc1A in E. coli and could show the

appearance of a polypeptide which binds specifically to the inverted

repeat of Tc1 .We have characterized the DNA binding further by

DNase I footprinting and observed protection between basepairs 3

and 30 relative to the end of the transposon. This suggested that the

terminal 6 nucleotides shared between most members of the Tc1

family (TACAGT) are not involved in sequence specific binding of the

transposase to the ends of the element. Mutation analysis of the

inverted repeat showed that the TAGATC sequence is indeed not

important for bindinq of Tc1A to the inverted repeat. Perhaps they

play a role in a later step in the transposition pathway. Purification

of the polypeptide used for these assays revealed that a (probably

proteolytic) derivative of Tc1A was responsible for the high-

affinity DNA binding activity. In contrast, the full-length Tc1A

protein has a very low affinity for the inverted repeat, which

suggests the presence of a domain in Tc1 A which regulates the DNA

binding potential. A similar result was obtained when we analyzed

Tc1 A DNA binding activity in nuclear extracts prepared from

induced transgenic animals. In a gel retardation assay we detected a

complex which migrates at a position expected for a smaller version

of Tc1A .We do not know what the significance of this observation

is, but one may speculate about the occurrence of a smaller version

of Tc1A which acts as a repressor in vivo. Analysis of carboxy-

terminal deletion mutants of Tc1A showed that the domain required

for site-specific binding is contained within the first 63 amino

acids. Thus, the coding information for inverted repeat binding is

mostly contained within the first exon of Tc1A .Furthermore, there

is a second DNA binding domain, because a construct in which the

first 70 amino acids of Tc1 A are absent, was previously shown to

contain a strong general DNA binding domain (Schukkink and

Plasterk, NAR 1990). In summary, we conclude that Tc1A is a

transposase and has two independent DNA binding domains, one N-

terminal for the binding to the inverted repeat of Tc1 and a second

non-specific DNA binding domain which possibly interacts with

target DNA in the transposition reaction.