Plant Molecular Biology 44: 91–98, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
Interaction of nuclear proteins with intrinsically curved DNA in a matrix
attachment region of a tobacco gene
Plant Molecular Biology Laboratory, Molecular Biology Department, National Institute of Bioscience and Human
Technology, AIST, MITI, Higashi 1-1, Tsukuba, Ibaraki 305-8566, Japan (e-mail: email@example.com)
Received 16 July 1999; accepted in revised form 23 May 2000
Key words: chitinase gene, curved DNA, nuclear matrix attachment region, nuclear protein
Two scaffold/matrix attachment regions (S/MARs), designated S/M I and S/M II, are located in the 5
region of the tobacco basic class I chitinase gene, CHN50. Structural analysis of these S/MARs showed that S/M II
contained an intrinsically curved DNA sequence that is located between −1786 and −1722 relative to the initiation
site of transcription. Electrophoretic mobility shift assays and southwestern blotting analysis were performed to
identify the tobacco nuclear proteins that bind speciﬁcally to this curved DNA. These experiments revealed that
nuclear proteins bound speciﬁcally to the curved DNA. Moreover, the nuclear proteins appeared to recognize the
overall structure of the intrinsically curved DNA, as distinct from binding to the DNA with sequence speciﬁcity.
Southwesternblottinganalysisshowed thatproteinsof 22, 24, 28 and 34 kDaboundspeciﬁcally to the curvedDNA.
The possible functions of the binding proteins and their relationship to previously identiﬁed nuclear proteins, such
as high-mobility-group proteins, are discussed.
Intrinsically curvedDNA is often found near function-
ally signiﬁcant regions of genomes, for example in
promoter regions (McAllister and Achberger, 1989),
at the origins of DNA replication (Zahn and Blattner,
1985; Snyder et al., 1986), in the DNA of centromeres
(Bechert et al., 1999), at sites of speciﬁc recombina-
tion of DNA in both prokaryotes (Hagermann, 1990)
and eukaryotes (Milot et al., 1992), and in scaffold
or matrix attachment regions (S/MARs) that anchor
chromatin to the nuclear scaffold or matrix (von Kries
et al., 1990; Hibino et al., 1993; Fukuda, 1999). Al-
though the biological effects of intrinsically curved
DNA are not fully understood, it has been proposed
that activation of transcription, illegitimate recom-
bination of DNA, replication of DNA (Hagermann,
Kiyama, 1994) are associated with such curvature. In
particular, a functional role for curved DNA in the
regulation of prokaryotic transcription has been sug-
gested by a number of studies (Bracco et al., 1989;
Collis et al., 1989; McAllister and Achberger, 1989;
Gartenberg and Crother, 1991; Lavigne et al., 1992).
In addition, evidence implicating DNA curvature in
the activation of eukaryotic transcription has been
presented (Kim et al., 1995; Ohyama, 1996).
It has been reported that intrinsic DNA curvature
facilitates the binding of certain proteins, such as
DNA topoisomerase I (Camilloni et al., 1991) and
II (Howard et al., 1991), several nuclear scaffold
proteins (Hibino et al., 1993), high mobility-group
proteins 1 and 2 (HMG-1/2) (Churchill et al., 1995),
and a histone-like nucleoid protein in Escherichiacoli,
which is known as H-NS (Atlung and Ingmer, 1997).
While HMG-1/2 stimulate the binding of certain tran-
scription factors to their speciﬁc target sites and can
enhance the activation of transcription (Zwilling et al.,
1995; Paull et al., 1996; Zappavigna et al., 1996),
H-NS appears to modulate the expression of a large
number of genes via a negative effect on transcrip-
tion (Atlung and Ingmer, 1997). In plants, intrinsically
curved DNA sequences have been found in the ribo-
somal RNA genes and to interact with HMG-related