*Institute of Molecular Evolutionary Genetics, Department of Biology, Pennsylvania State University;
and
Medical Research Council Molecular Haematology Unit, Institute of Molecular Medicine, University of Oxford;
and
Institute of Biological Anthropology, University of Oxford
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Abstract |
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Introduction |
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The mutational consequences of proximity to eukaryotic replication origins has thus far not been investigated, despite known differences in the replication fidelity of polymerases involved in replication initiation, strand elongation, and the gap filling which follows removal of RNA primers (Kunkel 1992
). The extent to which DNA replication fidelity is enhanced or compromised in proximity to a replication origin can, in theory, be investigated by analysis of polymorphism and divergence at sequences known to act as replication origins. This is because levels of polymorphism and divergence are both expected to be higher in regions experiencing a higher relative rate of neutral mutation. Prokaryotic oris, for example, show extensive sequence conservation between species (Sharp et al. 1989
; Kornberg and Baker 1991
, p. 534) and low interstrain diversity (Spangenberg, Montie, and Tummler 1998
), suggesting either high-fidelity replication/repair of these regions or selective constraint consistent with their essential role in DNA metabolism.
Characterization of replication origins in eukaryotes has advanced in recent years such that a significant number of candidate regions are now recognized. The best characterized eukaryotic oris are found in yeast and have been shown to comprise a number of modular domains (Marahrens and Stillman 1996
). In all cases, the characterized replication origins contain a conserved (but degenerate) short sequence domain, known to bind the origin recognition protein complex involved in controlling the timing and location of replication initiation, and a less well-conserved domain, often with a low inherent helical stability, believed to function as a so-called DNA unwinding element (DUE) (DePamphilis 1999
). Initiation of DNA replication begins when the DNA comprising the DUE unwinds and the
polymerase-primase complex synthesizes a RNA primer; DNA synthesis then proceeds bidirectionally from the DUE core (Kornberg and Baker 1991
).
Human replication origins have been shown to contain similar modular elements (Dobbs, Shaiu, and Benbow 1994
; Aladjem et al. 1998
) and DNA-protein binding analyses at one origin have been reported (Dimitrova et al. 1996
; Abdurashidova et al. 1998
). To date, the only genetically defined human replication origin is one found 5' of the human ß-globin locus. Preliminary analyses of this origin using a replication direction assay (a biochemical method that identifies the initiation region [IR] of an ori by detecting a switch in leading-strand synthesis) showed that replication proceeded bidirectionally from a genomic region approximately 2.0 kb in length, regardless of cell type (Kitsberg et al. 1993
). Deletion of this region in a Hemoglobin Lepore patient resulted in a reversal of strand synthesis polarity, suggesting that the ß-globin domain was being replicated by a different ori, located upstream of the ß-globin gene cluster. Subsequent investigation narrowed the defined IR to a 1.3-kb segment (which overlapped the same region identified by Kitsberg et al. 1993
) and showed that interaction of the IR with the upstream locus control region was required for origin activation (Aladjem et al. 1995
). Most recently, Aladjem et al. (1998)
have demonstrated that specific sequences in the vicinity of the initiation region (adjacent to but outside of the IR itself) are required for origin function. No analysis of replication-associated protein binding in this region has thus far been reported.
The ß-globin gene cluster is one of the most extensively investigated gene clusters in higher eukaryotes and represents an excellent candidate region in which to investigate patterns of polymorphism and divergence associated with proximity to a constitutive ori. At the time the replication origin was first identified, we were engaged in an investigation of polymorphism in the nearby human ß-globin locus (Fullerton et al. 1994
; Harding et al. 1997
), an analysis that overlapped the defined IR. We therefore extended our investigation, for a subset of the original sample, to include the whole of the IR and a significant length of 5'-flanking DNA. Our results suggest that a subregion of the replication origin initiation zone, composed of an AT-rich alternating purine-pyrimidine ((RY)n) repeat with DNA-unwinding capability, is subject to marked intraspecific and interspecific sequence divergence. Unexpectedly, the pattern of polymorphism at this complex repeat sequence differs substantially from variation observed at other human complex microsatellites, suggesting that nucleotide composition alone is unlikely to explain the observed hypervariability. Instead, a higher underlying rate of neutral nucleotide substitution, associated with the role of the (RY)n in replication initiation, is implicated.
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Materials and Methods |
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DNA Sequence Analysis
Bases 5829864374 of the HUMHBB GenBank sequence (accession number U01317) were amplified from genomic DNA and sequenced manually with Sequenase T7 DNA polymerase (United States Biochemical). The 6-kb region was amplified as two 3-kb fragments (sites 13009 and 30106076, respectively). DNA amplification and direct sequencing in both regions was conducted in the same manner, following methods for the analysis of the 3' fragment published previously (Fullerton et al. 1994
). Sequence haplotype linkage relationships were determined for the 3' fragment only. Primers for the amplification of the 5' fragment corresponded to positions 5829858322 and 6130761331 of the reference sequence. The sequences of primers used in the analysis of the entire 6-kb region are available on request. GenBank accession numbers for the IR sequence haplotypes are AF186606AF186620. The chimpanzee ß-globin sequence used for the interspecific comparison was that reported by Savatier et al. (1985)
(GenBank accession number X02345).
Estimating Genetic Diversity and Sequence Divergence
Genetic diversity in subregions of the 6-kb segment investigated was described in terms of per-nucleotide expected heterozygosity, or nucleotide diversity. Two estimates of nucleotide diversity were derived using the statistical analysis package DnaSP, version 3 (Rozas and Rozas 1999
). The first,
, is based on the observed number of polymorphic nucleotide sites or nucleotide and length (indel) changes observed in a sample of DNA sequences (Watterson 1975
). The second,
, measures the average pairwise sequence difference between any two random alleles in the sample investigated (Nei 1987
, p. 256). Standard errors for each estimate were calculated assuming no recombination between sites, which results in a more conservative value than assuming free recombination (Nei 1987
, pp. 255257). The two estimates were compared via Tajimas (1989)
D statistic. Values of D significantly different from 0 suggest that the two estimates of nucleotide diversity are discordant and hence that the observed variation is not consistent with the null hypothesis of neutrality. Significant differences in estimates of nucleotide diversity for particular sequence subregions were identified using the distribution of estimates generated from 10,000 independent coalescent simulations, assuming a neutral infinite-sites model without recombination and large constant population size (Hudson 1990
).
Comparison of levels of intraspecific polymorphism and interspecific divergence was performed according to Hudson, Kreitman, and Aguadé (1987)
(the HKA test), using DnaSPs Direct Mode feature. Nucleotide sequence differences between humans and chimpanzees were estimated from a human-chimpanzee sequence alignment generated using the SIM sequence alignment program (Huang, Hardison, and Miller 1990
), with match, mismatch, gap-open, and gap-extension penalties of 1, -0.5, 1, and 0.5, respectively. This alignment was identical to one originally reported by Savatier et al. (1985)
. Comparisons were performed using nucleotide site differences only and using nucleotide and length changes considered together.
Sliding-window plots of nucleotide diversity, , for the total sample and each of the population samples, as well as plots of the average number of nucleotide substitutions per site between populations, Dxy, and nucleotide divergence between humans and chimpanzees (calculated as the average proportion of nucleotide differences between species, K [Nei 1987
, p. 276]), were generated using DnaSP. In all cases, parameter values were calculated for 100-bp windows placed at 5-bp intervals. Length polymorphism/divergence was disregarded in each of these analyses.
Pairwise sequence differences for the (RY)n repeat sequence and seven additional human complex microsatellites were calculated using alignments of unique sequence haplotypes found at each locus. Sequence data for five loci were obtained from GenBank: RNU2 (Liao and Weiner 1995
; accession numbers U57504U57614), Mfd 59 (Garza and Freimer 1996
; U48313U48320), DQCAR (Macaubas et al. 1997
; U96944U96962), and DQCARII and G51152 (Lin et al. 1998
; AF042291 AF042316). Sequences for the other two loci, MIB (Grimaldi and Crouau-Roy 1997
) and DRB1 (Bergström et al. 1999
), were inputted manually using data presented in the published reports. Alignments were reconstructed to reflect those provided by the original authors, where available. All other sequences, including the (RY)n region, were manually aligned.
Calculation of Helical Stability
Helical stability in the 6-kb region surveyed for polymorphism was determined using the computer program THERMODYN (courtesy of D. Kowalski). The program uses experimentally determined thermodynamic parameters to calculate the free energy difference (G) between the duplex and single-stranded states for multiple overlapping segments or "windows" of a particular DNA sequence (Natale, Schubert, and Kowalski 1992
). Values of
G (in kcal/mol) were calculated for 100-bp windows placed at 5-bp intervals along the 6-kb consensus (GenBank) sequence, assuming a temperature of 37°C and an ionic strength of 10 mM.
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Results |
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DNA Helical Stability in the ß-Globin Replication Initiation Zone
The striking preservation of the (RY)n repeat sequence structure in the face of high levels of substitutional polymorphism and divergence suggests that its structure may have a functional significance, possibly in the context of replication initiation. While little is currently known about specific regulatory sequences associated with human replication origins, it is possible to assess the helical stability of duplex DNA by a consideration of base-stacking interactions among nucleotides in a given sequence (Natale, Schubert, and Kowalski 1992
). Using a computer program designed for this purpose (see Materials and Methods), we were able to investigate the nature of DNA-unwinding capability in the ß-globin replication initiation region.
As shown in figure 5, a region of pronounced helical instability is present in the defined IR, which coincides with the hypervariable (RY)n sequence. This suggests that the (RY)n sequence constitutes the DUE of the replication origin initiation region, i.e., the place at which DNA unwinding begins in preparation for initial strand synthesis. Similar investigation of the DNA-unwinding profile of African and European sequence haplotypes suggests that the observed polymorphisms, which retain the alternating (RY)n structure, have no detectable effect on DNA helical stability (data not shown). This (indirect) evidence supports the assumption that the observed polymorphisms persist in human populations because they have no appreciable deleterious effect on the function of the ß-globin ori. As the primary unwinding domain of an origins IR is likely to lie in close proximity to the start site of replicative synthesis, the hypervariability of the ß-globin DUE also suggests that only enzymatic interactions involved in the earliest stages of replication initiation are error-prone.
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Discussion |
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The polymorphisms and substitutions observed in the ß-globin replication origin IR represent mutations that have eluded both mismatch repair and the effects of drift and purifying selection. The presence of allelic variants at significant frequencies in human populations (data reported here and in Harding et al. [1997
]) suggest that the observed polymorphisms are not seriously deleterious, although possible functional effects of the variation clearly merit direct investigation. The fact that none of the observed variants disrupt the helical stability profile of the DUE is consistent both with the apparent neutrality of the variation and with the influence of selective constraint in determining the position and type of polymorphism found in the DUE. If the effects of constraint are significant, the observed sequence diversity may not reflect the full spectrum of mutational events that arise at the ß-globin replication origin. Investigation of de novo germ line or somatic mutation in the same region will provide relevant information regarding the mutational basis of the observed variation and should clarify the extent to which mutation rates vary within and around the replication origin initiation region.
The accumulation of polymorphism in the replication origin DUE, which is likely to lie at or near the start site of replication (Bielinksy and Gerbi 1998
), suggests that DNA polymerase fidelity and/or DNA repair associated with replication initiation at the ß-globin ori may be compromised. These findings are consistent with observed differences in in vitro DNA polymerase error rates, which have suggested that different phases of replicative DNA synthesis (e.g., initiation vs. strand elongation) may be subject to different rates of mutation (Kunkel 1992
). ß polymerase, for example, is known to play a role in small gap repair and hence may be involved in the excision of the RNA primer (and the first 2550 nt of DNA synthesized by
polymerase) at the replication initiation start site (Linn 1991
). However, the enzymes putative role in DNA synthesis at replication origins is less compelling in this context than is its known involvement in a form of DNA replication error known as dislocation mutagenesis (Kunkel and Alexander 1986
). Dislocation mutagenesis results from transient template-primer slippage, in which the template slips with respect to the enzyme, a correct nucleotide is incorporated, and the template then realigns prior to continued synthesis, resulting in an apparent misincorporation event. This form of mutagenesis, which results in base substitutions and 1-nt frameshifts at distinct hot spots in vitro (Kunkel and Soni 1988
), is consistent with many (if not all) of the polymorphisms observed in the ß-globin (RY)n repeat region (fig. 2
). Moreover, dislocation mutagenesis is a unique feature of ß polymerase; no other eukaryotic DNA polymerases generate such errors in vitro (Roberts and Kunkel 1996
).
This unusual feature of the ß polymerase enzyme, along with its possible role in the replication process, suggests that errors associated either with small gap filling following RNA primer excision or base excision repair are responsible for the observed hypervariability of the ß-globin replication origin IR. If so, these observations imply that eukaryotic replication origins may in general be subject to higher-than-average rates of within- and between-species sequence divergence. More specifically, our findings predict that sequences involved in DNA unwinding at eukaryotic replication origins, which lie in close proximity to the RNA primer synthesis site, may be particularly susceptible to nucleotide substitution and small length changes.
Although no other investigation of origin-associated polymorphic variation has thus far been reported, available evidence from other eukaryotic replication origins is consistent with these predictions. There is, for example, a well-known lack of sequence conservation among replication origins in the genome of the yeast Saccharomyces cerevisiae (Broach et al. 1983
), which contrasts markedly with the extensive sequence similarity observed among prokaryotic oris (Kornberg and Baker 1991
, p. 534). Of the six replication origins which have been well-characterized in this species (Theis et al. 1999
), only one of the two main modular elements commonly present, domain A, is conserved, and within this domain, conservation is restricted principally to a single essential 11-bp motif (Marahrens and Stillman 1996
). Extensive differences among oris are observed in the B domain, which encompasses an easily unwound sequence suggested to function as a DUE (Natale, Umek, and Kowalski 1993
). Conservation of domain A and element B1 of the B domain (but no other B-domain elements) has also been observed among homologous origins found in distinct Saccharomyces species (Theis et al. 1999
). However, no sequence conservation exists between replication origins found in budding and fission yeast (Clyne and Kelly 1995
).
One study has reported that in humans, the intergenic spacer of the rRNA repeat accumulates variation at high rates (Gonzalez and Sylvester 1995
), a feature of rDNA found in a wide variety of organisms. The human intergenic spacer has also been shown in independent investigations to contain one or more initiation zones for DNA replication (Little, Platt, and Schildkraut 1993
; Gencheva, Anachkova, and Russev 1996
). The extent to which the reported variation coincides with the mapped initiation zone(s) has not been investigated. A pilot survey of variation in the vicinity of two other human replication origins suggests that these too may experience higher-than-average levels of nucleotide and length polymorphism (unpublished data).
The effects of selective constraint on regions subject to high rates of sequence turnover are reflected in the composition and genomic location of eukaryotic replication origins. If constraint acts primarily on DNA secondary structure (e.g., unwinding capacity), higher underlying rates of polymorphism and divergence in replication origin IRs are expected to favor conservation of general modular features rather than specific sequence motifs. This feature of replication origins is well known (Dobbs, Shaiu, and Benbow 1994
; Marahrens and Stillman 1996
). Similarly, to minimize the likelihood of deleterious mutations disrupting origin function, important regulatory sequences in the ori (which rely on specific base pair interactions) are more likely to lie at a distance from the start site of replication initiation. The best characterized replication origins in budding yeast occupy genomic regions of 100200 bp in length, consistent with moderately broad positioning of regulatory elements; origins in higher eukaryotes are less well defined (Gilbert 1998
). Indeed, the involvement of the far upstream (located 50 kb 5') locus control region in stimulating replication at the ß-globin origin suggests that cis-acting elements may be found at considerable distances from the start site of initiation (Aladjem et al. 1995
). Finally, the hypervariability of replication origin initiation zones suggests that they should be confined to noncoding flanking and intervening sequences so that the integrity of adjacent coding and regulatory regions is preserved. Consistent with this prediction, the majority of known eukaryotic replication origins are, like the ß-globin ori, located in intergenic DNA (Brewer 1994
).
A predisposition to higher-than-average rates of nucleotide substitution would, at first glance, appear to be a significant disadvantage for genomic regions that have an important role to play in DNA replication. The modular nature of eukaryotic replication origins, and at least partial reliance on elements whose function is determined by secondary structure rather than by nucleotide composition, must contribute to the tolerance of these regions to high rates of sequence turnover. Unexpectedly, however, high levels of polymorphism and divergence relative to adjacent noncoding regions at the ß-globin ori suggest that replication origins are subject to higher rates of mutation than nonfunctional DNA. Why relaxed replication fidelity is associated with replication initiation and why it persists in the face of selective pressures which should, on balance, favor high-fidelity synthesis is unclear. The finding of significant sequence diversity in close proximity to an important (and, until relatively recently, unrecognized) functional domain in the ß-globin gene cluster illustrates the complexity of forces with the potential to influence rates of spontaneous mutation in eukaryotic genomes. Investigation of population variation and interspecific divergence in this and other origin regions promises to further illuminate our understanding of both basic mutational mechanisms and replication origin biology.
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Acknowledgements |
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Footnotes |
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1 Abbreviations: DUE, DNA unwinding element; IR, initiation region; ori, origin of replication.
2 Keywords: globin,
DNA replication,
polymerase fidelity,
mutation,
polymorphism,
human.
3 Address for correspondence and reprints: Stephanie M. Fullerton, Institute of Molecular Evolutionary Genetics, Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802. E-mail: smf15{at}psu.edu
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