“Airborne” Holliday Junctions in the human genome transposed by mobile elements

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“Airborne” Holliday Junctions in the human genome transposed by mobile elements

Transposable Holliday Junctions in the human genome

Purpose: Holliday Junctions (HJ) constitute important intermediate structures for many cell functions as in DNA recombination and DNA repair. These structures result from different formations of a common sequence pattern, with a 3 nucleotide core motif and a total length of 10 noucleotides. In this study, we explored whether this degenerate sequence pattern coincides with retroelement sequences and more specifically with the active and inactive retrotransposable element families ALU, LINE, SVA and HERV in the human genome.

Methods and Results: A database search was conducted in order to identify the variations of the different forms of the sequence d(CCnnnN6N7N8GG) . This conserved short sequence was found in six different forms. Based on the six motifs , we identified and located their coordinates in every human chromosome sequence using the R 2-15-0 console. All identified sequences were verified, using the FASTA format in the “HapMap Project” database. The online RepeatMasker software was employed to assign the respective sequences of the implicated transposable elements. Holliday Junctions are found in high percentages, up to 50%, in all main retroelements families. These structures are present in both active and inactive transposable element's sequences. Transposable elements exhibit a high specificity for particular HJ sequence variations and certain active elements are invariably transposed carrying these particular HJ.

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Conclusion: This is the first report to localize Holliday Junctions on transposable elements. Specific human HJ sequences have a preference for particular types of mobile elements. Active and evolutionary younger mobile elements have almost 100% linkage with certain Holliday Junction sequences.

Key words: Holiday Junctions, retroelements, recombination,

Abbreviations: HJ, RE

BACKGROUND

Holliday junction (HJ) is essentially a four-way junction , a structure in which four double-helical DNA molecules are connected by mutually exchanging component strands®. The structure is named after Robin Holliday who described it in 1964 , when he identified a particular type of genetic information exchange in fungi also known as homologous recombination. The Holliday Junctions are conserved structures found in all living organisms, from prokaryotes, to mammals and have been recognized as important intermediates involved in several cellular processes, such as DNA recombination.

Homologous recombination is engaged, in a wide variety of cellular processes. Originally described as a way to generate genetic variety with new combinations of genes, has now been proved to be essential for the following: 1) the viral integration into hosts, 2) the maintenance of genomic stability through the repair of DNA damage, 3) the reactivation of the stalled replicative fork, and 4) the even separation of homologous chromosomes during cellular division. Any mistake in the recombination process, results in increased mutagenesis, meiotic/mitotic aneuploidy and genomic instability. Effects that have been associated with the occurrence of numerous disorders. A large number of single crystal Holliday structures has been discovered in recent years, demonstrating the variability in their conformation . Data concerning the intrinsic conformation of the junction sequences show, variation in interactions with drugs and ions in respect to base modifications and sequence variations that affect the accessibility of the structure to proteins. Basically, the junction appears to be dependent on a common trinucleotide motif in 6th, 7th and 8th position of the 10nt sequence d(CCnnnN6N7C8GG), forming strong hydrogen bonds , which stabilize the structure. HJs have been in the spotlight ever since they were introduced by Robin Holliday when the general structure was established, through the first experimental models and found to be dependent on the type and concentration of specific ions. The HJs adopt a closed conformation in high ion concentration in contrast to an open conformation in low ion concentration which gives to the structure the ability to move along homologous DNA chains (branch migration). The dynamics of the recombination events in the human genome are related to HJs but also to the presence of DNA low copy repeats (LCRs). The vast majority of the repeated DNA sequences is composed of various transposable element families acquired through evolution in the human genome from other species but also of primate and human specific elements.

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Almost 45% of the human genome is composed of DNA related to transposable elements (TEs) and has the ability to relocate in the chromosomes. Approximately 90% of these DNA sequences are related to retrotransposable elements, but only a minority of those is active and can relocate. Active retroelements are mediated by RNA transcription, reverse transcription to cDNA and integration to a new site, either intragenic, or intergenic. The retrotransposable elements are roughly discriminated by their two constitutional characteristics in: 1) LTR and non-LTR and 2) autonomous or non-autonomous. Human endogenous retroviruses (HERVS) possess LTR in contrast to the other retroelements and are therefore autonomous. LINE 1 is also autonomous but non-LTR, representing almost 20% of the retroelement related sequences in the genome. LINE 1 has in addition the ability to mobilize non autonomous retroelements and drive them to the ribosomes for processing. Under normal conditions the retroelements are controlled and regulated by DNA methylation, non coding RNAs and suppressing proteins. Any escape from the controlling agents or deregulation may have an effect on the cellular state of differentiation and division. Exogenously introduced retroelements have been found to alter cellular phenotypes and interfere with proliferation. The perpetual transcription and integration cycles of the active retroelements reshapes the genome through methylation fluctuations during meiosis and mitosis. A number of single gene disorders are caused by novel retroelement insertions, and numerous chromosomal aberrations are the result of recombinations involving retroelements. On top of the above, over or under expression of endogenous retroviruses is found in autoimmune disorders, and many cancers of the reproductive or non-reproductive cells and tissues.

This study was conducted in order to explore the ability of HJs to be mobilized and transposed by TEs using their ability to function as vectors in the human genome. The deeper understanding of the intermediates in the formation of recombination hotspots, will highlight evolutionary issues of the human genome. Furthermore will provide evidence for the genetic background underlying human acquired and inherited disorders.

R esults

HJ Sequence motif identification

After extensive search in selected databases containing bio data we found out that Holliday structures usually consist of inverted repeats ( IR ) of a conserved dekanoukleotide form having the d [CCnnnN(6)N[7]C[8]GG] sequence structure. The crystal structures of Holliday Junctions longer identified by inverted repeats (IR sequences) in the 10-mer d (CCnnnN6N7C8GG) which contain the common core ACC (ACC-triplet) and may be formed by (i) replacing terminal bases GC by TA, (ii) replacing cytosine to methylated cytosine at position N8, (iii) replacing inosine bases by 2- aminopurine and brominated accordings to the positions N6N7N8 and (iv) in the presence of divalent cations including magnesium, calcium and strontium. These structures confirmed that (i) the basic core (but not absolutely necessary) to generate HJ is ACC, (ii) the interaction of the amino group of cytosine C8 with phosphate groups in cross-over points are important not only for the formation of HJ but also for determining the geometry of the same, and (iii) the presence of divalent cations contributes to the final geometry of the nodes. We have seen that the existence of the ACC core to the presaid 10 mer have been prerequisite for the creation of Holliday Junctions but certainly not necessary. After conducting several surveys it is commonly known that the existence of different triplets in these positions lead to the formation of Holliday junctions. Experimental studies in the decamer sequence pattern d (CCnnnN6N7C8GG) (NNC wherein any of the 4 nucleotides, nnn the inverted repeats of NNC) showed the existence of three different triplets that form HJ under specific circumstances. We emphasize under specific circumstances, because unlike as the ACC core that forms only HJ, the existence of three other triplets does not lead only to the formation of HJ structures but also to A-DNA and B-DNA structures. These are the socalled amphimorphic sequences. More specifically trinucleotides N6N7N8 = ACC, GCC, ATC, CCC and their respective larger sequences may under suitable conditions form HJ. The GCC-core sequence forms HJ in the presence of Ca2 + (calcium cations) while it displays B-DNA features in the presence of Mg2 + (magnesium cations). Furthermore the ATC-core sequence forms HJ such as B-DNA only in the presence of Ca2 +, which adopts the closed (stacked-X) configuration at high ion concentrations. In smaller ion concentrations it adopts open (open-X) configuration which permits migration (branch migration) along DNA heliqs. Finally, the CCC-core sequence forms HJ only in the presence of Ca2 + in high concentration, while A -DNA structure in a low concentration of Ca2 + at this time. After comparing the sequences containing these 4 different cores we obtained the general features of the sequences that form Holliday Junctions. Therefore the sequences identified to form HJ, are :

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1. d(CCGGGACCGG), 2. d(CCGGΤACCGG), 3. . d(TCGGTACCGA), 4.

d(CCGGGCCCGG), 5. d(CCGGCGCCGG), 6. d(CCGATATCGG).

a/a

HJ

ALUs

LINE-1

ERVLMalRs

ERV-

classI

DNA

elements

SVA

1

d(CCGGGACCGG)

8.35%

────

────

5.74%

────

2

d(CCGGTACCGG)

────

36.61%

────

────

29.52%

3

d(TCGGTACCGA)

────

10.78%

25%

────

11.52%

4

d(CCGGGCCCGG)

33.15%

────

────

────

────

5

d(CCGGCGCCGG)

────

────

────

46.71%

────

6

d(CCGATATCGG)

48.9%

────

────

────

────

Table 1. Probabilities of each particular HJ sequence to reside in a particular retroelement. One to 3 of the six HJ sequences can be found in each particular Retroelement type with the exception of HJ sequences 3 and 4 which have absolute specificity for ALUs and ERV-class I and are not found in other REs.

The percentages represent the probabilities in a distance of 50nt to the 5’ prime end of the HJ and 50nt to the 3’ prime end (graph).

""Με a → d(CCGGGACCGG), b→ d(CCGGTACCGG), c→ d(TCGGTACCGA), d→ d(CCGGGCCCGG), e→ d(CCGGCGCCGG), f→ d(CCGATATCGG) συμβολίζουμε τις αλληλουχίες που σχηματίζουν HJ

The table shows general features of the sequences that form HJ. Each Hj sequence shows preference for different type of retroelement every time. In some cases, the specificity is up to 100% , eg the CCG-core HJ sequence has nearly 100% specificity with HERV-H retrovirus.

After processing all data and especially after recognition and identification of specific types that «carry» HJ, HJ proved to exist in active and inactive, evolutionary old and new retroelements, which are listed in evolutionary trees.

""

ALUs: AluJo, AluJ, AluSc, AluSx, AluSq, AluSz6, AluSq4, AluSx3, AluY, AluYc1, AluYh9, AluYb8, AluYi6 . Τα ρετρομεταθετά με κÏŒκκινο χρÏŽμα είναι active.

""

LINE1: L1MA1, L1MA2, L1MA4, L1P1, L1P2, L1P3, L1P4, L1PA15-16, L1PA10, L1HS

""

HERVs: HERVE, HERVK, ERVH, HERVL, HERV9, THE1

Human and Pan Troglodytes specific retroelements

""

SVAs: SVA_A, SVA_B, SVA_C, SVA_D, SVA_E, SVA_F

From the retroelement evolutionary trees it becomes evident that HJ s are present in cis in both evolutionary young and old elements and in active as well as in inactive. The SVA family is the only exception with HJsequence found in the youngest member , SVA_F.

Discussion

Our results demonstrate that HJs are mobile using retroelemets as vectors to disperse recombination spots throughout the genome. The existence of mobile holiday junctions in cis with retroelements, is a novel finding of this study demonstrating the synergistic and adaptive nature of sequences related to genomic rearrangements, found in almost all types of meiotic and mitotic recombinations. On top of the mobility and diaspora of HJs, with retrotroelements as their effective carriers, we also found a specificity of each and every HJ sequence with particular active and inactive retroelements. Both the above indicate that during the course of evolution diverse sequences of HJs have been hosted by certain retroelements to function as a “recombination unit” within chromosomes.

All three main recombination types existing, Homologous Recombination, ΝonHomologous Recombination and Replicative Recombination are utilizing HJs to perform in a variety of cells and tissues. Our findings point to a dispersing mechanism that reallocates all the important HJ sequences in the genome in order to generate novel recombination events but on preselected hotspot retroelements sequences.

Homologous recombination is one of the most fundamental mechanisms in nature, being responsible for meiotic crossover, integration of transferred DNA into host genomes and DNA repair in response to DNA double-strand breaks (DSBs). Although the detailed mechanism remains still elusive, they are probably common to all cells, and have also in common HJ branch migration. Homologous recombination is a type of genetic rearrangement that occurs through the breakage and rejoining of DNA molecules within a stretch of identical or very similar sequences. A common mechanism that allows the self-repair of DNA that has been damaged on both strands of double helix and can fix lesions that occur in almost every round of DNA replication. It is also necessary for the proper and accurate segregation of chromosomes during meiosis.

Non-homologous recombination also plays a central role in biology of the mammalian systems. For example, the development of metastatic tumors appears to involve the accumulation of a wide variety of NHR-related altered chromosomal patterns. Nonhomologous recombination occurs in regions where no large-scale sequence similarity is apparent, e.g. translocations between different chromosomes or deletions that remove several genes along a chromosome. However, short sequence similarity is found at the breakpoints where the procedure takes place and is also dependent on the presence of HJs. Furthermore NHR has been found to underlie meiotic recombination that result in human germ line chromosomal disorders.

Replicative recombination is a type of recombination which generates a new copy of a DNA segment. Many transposable elements (TEs) use a process of replicative recombination to generate a new copy of the transposable element at a new location. These elements usually propagate themselves in the genome by a repeated copy and paste pattern creating novel integration sites, novel gene exons and extensive genomic alterations also involving HJs.

Evolution in all genomes is driven by recombination, and permits meiotic and mitotic changes that accumulate overtime. The gradual accumulation of genetic alterations over a long period of time in reproductive or somatic cells, would result in a large-scale reformulation of the nucleotide sequence of the genome, and to extensive restructuring. The inert ability of the genome to create and exploit novel properties through recombination, would have not been present, and the evolutionary potential of the genome would have been restricted in the absence of widely interspersed HJs.

Genome stability on the other hand is characterized by the ability of the genome to proceed to DNA damage repair, to avoid loss of heterozygosity, and permit chromosome rearrangements without any burden to the meaningful DNA content. These powerful but counter dependent qualities of the genome are both evident in almost all cellular processes form meiosis to mitosis, present at germ line or somatic cell divisions. Mitotic recombination is a rare phenomenon per cell division in healthy cells, but is more common in destabilized cancer cells. In contrast meiotic recombination is more frequent in reproductive germ cells and is the basis for novel rearrangements in subsequent generations of human individuals. Both processes either mitotic or meiotic have the ability to impose reciprocal crossovers and convert genes involved in the recombination hot spots.

This reviving genome restructuring is at the same time, the cause and the effect of plasticity modulated by the coexistence of retroelements and HJs, which by close collaboration and interplay seem to be fundamental as they combine properties and join forces.

Adverse consequences of failures in the close collaboration between REs and HJs or the inability of the cells to control their potent joint properties, may lead to cancer, unbalanced translocations, mosaicism and other defects. Although they have been working closely in mammalian and human evolution to create stability, diversity and plasticity they may occasionally underlie clinical defects.