According to a primary structure of consensus sequence of γ-satellite DNA [11
], there are three GlaI recognition sites 5'-A(5mC)GT-3'/3'-TG(5mC)A-3' within monomeric DNA in positions 61, 121 and 207. Patterns of a total DNA hydrolysis with AspS9I and BstSCI have shown that products of γ-satellite DNA digestion have a molar concentration at least 20 times more than a concentration of LINE1 repeats digestions [7
]. Because of such difference in DNA concentration we have used a total mouse DNA preparation in study of γ-satellite DNA cleavage with GlaI. The obtained cleavage pattern corresponds to a limited hydrolysis of γ-satellite DNA with GlaI due to mutations of ACGT sites and undermethylation of CG dinucleotides.
Results of double digestion of γ-satellite DNA with GlaI and restriction endonucleases AcsI, Bst2UI, BstF5I and FatI have confirmed positions of enzymes' recognition sites and locations of small (60 bp, 86 bp and 88 bp) and intermediate (146 bp, 148 bp and 174 bp) GlaI digestion fragments in monomeric γ-satellite DNA.
Hydrolysis of γ-satellite DNA with HpyCH4IV and SspI has provided similar patterns of evenly distributed DNA fragments, which are monomers, dimers, trimers, etc. (Figure ). However, the reasons of such specific digestion of γ-satellite DNA with these restriction enzymes are different. Incomplete hydrolysis of γ-satellite DNA with SspI is explained by mutation of the enzyme recognition sequence [7
], whereas HpyCH4IV cleaves γ-satellite DNA due to undermethylation of ACGT site in position 207 (Table ).
Consensus sequences of γ-satellite DNA fragments obtained in our work (Additional file 1
) and published earlier [11
] are coinciding. Surprisingly, a consensus sequence, which has been determined for 30 monomeric γ-satellite DNA fragments sequenced in the work of Vissel and Choo (Additional file 2
), differs in two positions from a known one [11
] (missed A nucleotides in positions 68 and 188).
Vissel and Choo cleaved γ-satellite DNA with MnlI and determined a primary structure of obtained monomers [13
]. Earlier Horz and Altenburger [11
] studied MnlI digestion of γ-satellite DNA and showed that methylation of CG dinucleotide in position 74 blocks DNA hydrolysis. So, Vissel and Choo isolated only those DNA monomers, which were unmethylated in position 74 in two neighbouring MnlI sites. Besides a distinct consensus sequence, a detailed analysis of these monomers alignment (Additional file 2
) has revealed a different distribution of CG dinucleotide mutations in several positions of γ-satellite DNA (see Table ).
In our study we have observed 18,4%, 25,5% and 0% mutations of CG dinucleotide in positions 62, 122 and 208, respectively (Table ). Data of Vissel and Choo show that all 30 sequenced fragments don't have mutations in position 74 and have only one mutation of CG dinucleotide in position 177. On the other hand, there are 20% mutations of CG dinucleotide in position 208. An absence of CG dinucleotide mutations in position 74 may be explained, at least partially, by overlapping position 75 of this dinucleotide with MnlI recognition sequence. A discrepancy of CG dinucleotides mutations in position 208 in two works is a consequence of either a very special selection of DNA fragments in a work of Vissel and Choo (see above) or different sources of DNA (a mouse liver in our work and a cell line in the work of Vissel and Choo). Nevertheless, both works demonstrate maximum CG dinucleotides mutations in position 16 (around 30%) and in position 122 (about 26%). Moreover, there is a similar distribution of mutations in these CG dinucleotides: mostly mutated G in position 123 and roughly equal mutated C and G in positions 16 and 17.
In this work we have confirmed data on large proportion of γ-satellite DNA in a total mouse DNA preparation [7
]. Vissel and Choo isolated γ-satellite DNA from a total DNA and studied its structure. According to their data γ-satellite DNA is organized into long uninterrupted arrays of between 1,000 monomers and greater than 8,000 monomers, likely, located near chromosome centromers [13
So, γ-satellite DNA is a prolonged DNA and constitutes a significant part of total DNA. However, we could not find peaks of 234 bp and 468 bp in diagrams of genomic DNA digestion with restriction enzymes, which have one recognition site in γ-satellite DNA monomer [7
]. In fact, the most of γ-satellite DNA is not presented in the known databases [7
]. Nevertheless, in genomic database there are small blocks of sequenced γ-satellite DNA, which contain 10–140 monomers [14
]. DNA sequences of γ-satellite monomers from two such blocks, which are located in chromosomes 3 and 9, have been aligned in this work (Additional files 3
, respectively). Consensus sequences of γ-satellite DNA of these two blocks are almost identical and differ in nucleotides in positions 18 and 179. However, both of them have three identical differences from consensus sequence of arrayed γ-satellite DNA [[11
] and this work]: A instead of G in positions 123 and 202, T instead of A in position 233. Besides, CG dinucleotide in position 178 and in position 17 has become CA dinucleotide in consensus sequences of γ-satellite DNA in chromosomes 9 and 3, respectively. A change of G for A in position 123 destroys one of three ACGT sites and consensus sequence of γ-satellite DNA in both blocks of monomers contains only two GlaI recognition sites in positions 61 and 207.
A comparison of Table data between rows 1, 2 and 3, 4 shows a significant difference of CG dinucleotide mutations in γ-satellite DNA from arrays and in small blocks of monomers. We can see more mutation in all 8 positions of CG dinucleotides in the case of blocks of γ-satellite DNA monomers if compare to CG dinucleotides in arrayed γ-satellite DNA. Table shows that in arrayed γ-satellite DNA there is at least one conservative CG dinucleotide, whereas γ-satellite DNA from blocks of monomers doesn't have a conservative CG dinucleotide in any positions.
Absence of mutations [this work] and 50% methylation [[11
] and this work] of CG dinucleotide in position 208 allow to say about important role of this CG dinucleotide methylation/demethylation in function of arrayed γ-satellite DNA from mouse liver.