The orientation bias of Chi sequences is a general tendency of G-rich oligomers
Introduction
Chi sequences (5′-GCTGGTGG-3′) are cis-acting DNA elements that enhance recombination promoted by the RecBCD pathway in E. coli (Smith et al., 1995). The Chi sequence attenuates the activity of 3′-to-5′ exonucleases and activates a weaker 5′-to-3′ exonuclease but does not affect the helicase activity of RecBCD (Dixon and Kowalczykowski, 1991). It has been proposed that the orientation of the Chi sequences plays an important role in the reconstruction of arrested replication forks (Cox, 1998, Horiuchi and Fujimura, 1995, Kuzminov, 1995). After replication fork arrest and the introduction from the double-stranded (ds) break, RecBCD enters the ds DNA molecule and degrades it into a single-stranded (ss) DNA with a 3′ -OH end and oligonucleotides (Michel, 2000, Seigneur et al., 1998). When the enzyme meets a properly oriented Chi sequence, its exonuclease activity is suppressed, and the helicase activity creates ss DNA tails, which are then used by RecA and ss DNA-binding proteins to form a D-loop with intact sequences having homology with the ss tails (Marians, 2000). On the D-loop molecule, a new replication fork is then reconstructed.
The whole genome sequence project (Blattner et al., 1997, Itoh et al., 1999) of E. coli revealed that 75% of all Chi sequences are oriented in the direction of DNA replication (Eggleston and West, 1997). These properties of RecBCD and Chi sequences have led to the widely accepted notion that Chi sequences are over-represented on the leading strand to direct the activity of RecBCD enzymes toward OriC (Blattner et al., 1997, Cox, 1998, Tracy et al., 1997b). The analogs of Chi sequences in E. coli (ChiEC) that are also G-rich (El Karoui et al., 1999) are found in B. subtilis (ChiBS 5′-AGCGG-3′), H. influenzae (ChiHI 5′-GNTGGTGG-3’ and 5′-G[C/G]TGGAGG-3′), and Lactococcus lactis (ChiLL 5′-GCGCGTG-3′) (Biswas et al., 1995, Chédin et al., 1998, Sourice et al., 1998). Every Chi sequence found is known to have a role in attenuating 5′-to-3′ exonuclease activity, and ChiLL has already been identified as having recombinase activity (El Karoui et al., 1998). In addition, research has shown that the processing of ds DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 3′-terminated ss tail, a universal intermediate of the recombination process in B. subtilis (Chédin et al., 2000). While these functions of RecBCD (in H. influenzae), AddAB (in B. subtilis), and RexAB (in L. lactis) are conserved, the structures of these proteins are not (El Karoui et al., 1999).
The GC skew, which represents a bias of G over C content in one of the duplex DNA strands, is clearly visible in the leading strands of the chromosomes of E. coli and B. subtilis but not as evident on that of H. influenzae (Blattner et al., 1997, Francino and Ochman, 1997, Lobry, 1996, Mrázek and Karlin, 1998). Regions on the DNA containing a shift in GC content (hereafter referred to as the shift point of the GC skew) often correlate with the locations of the replication origin and terminus. Several explanations for the cause of GC skew have been suggested:
- 1.
Asymmetric DNA replication between the leading and lagging strands could result in strand-dependent mutation patterns (Beletskii and Bhagwat, 1998, Francino and Ochman, 1997, Lobry, 1996).
- 2.
The requirement that genes that encode proteins cause biases in the base composition at codon positions 1 and 2 on the sense strand (McLean et al., 1998, Mrázek and Karlin, 1998, Romero et al., 2000).
It is proposed that 75% of the Chi sequences are oriented for the replication mechanism. However, it is also known that several octamer sequences switch their preferred orientation at the replication origin of several genomes (Salzberg et al., 1998). Species in which GC skew is clearly observed tend to contain many biased oligomers. In this work, we hypothesized that the Chi sequence reflects the tendency of the genome exemplified by the GC skew, so the orientation bias of the Chi sequence did not result from its function in replication mechanism. Under this hypothesis, the orientations of all G-rich oligomers should be similar to that of the Chi sequence. The genomes of E. coli, B. subtilis, and H. influenzae were analyzed (Fig. 1) for this purpose.
Section snippets
Materials and methods
The complete genome sequences of the three prokaryotes were downloaded from the GenBank database (ftp://ncbi.nlm.nih.gov/genbank/genomes/). GC skew was estimated from the formula (C−G)/(C+G) with non-overlapping windows whose lengths were fixed at 10 kb. The value of the GC skew is positive when the number of Cs is more than that of Gs in the same window, or vice versa. Transition of GC skew values over the entire genome was then graphed.
The orientation fraction of oligomers was defined as the
Results
The orientation fractions of hexamers, heptamers and octamers on the E. coli genome were calculated. The distributions of plots and their averages shift to a higher fraction as the G content of oligomers increases (Fig. 3). This shift must be caused by the asymmetry in G content between both strands, and this corresponds to the GC skew. Although Chi sequences have higher orientation fractions than the averages, many other octamers have even higher fractions (Fig. 3c). The orientation fractions
Orientational bias of Chi sequence depends on the bias of Guanines
In summary, the GC skew and the orientation bias of oligomers are strongly correlated, and the orientation bias of Chi sequences does not significantly differ from those of other G-rich oligomers (Fig. 6). While the Chi sequences of E. coli were oriented with a slight bias, many other oligomers showed a greater orientation bias. The species that showed a clear GC skew (E. coli and B. subtilis) also showed a clearly biased orientation pattern, whereas species with an unclear GC skew (H.
Acknowledgements
We thank Ichizo Kobayashi, Takashi Horiuchi, Naofumi Handa, Hirotada Mori and Stephen C. Kowalczykowski for helpful suggestions during this work. Furthermore, we would like to acknowledge the technical advice by Hiroaki Sakai, Takanori Washio, Kenji Yotsutani, Yumi Kawamura, Naota Ishikawa and Takehito Mogami. This work was supported in part by the Japan Science and Technology Corporation and a Grant-in-Aid for Scientific Research on Priority Areas ‘Genome Science’ from The Ministry of
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