Examining The Mechanisms Behind Structural Dna Exchanges Biology Essay

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Any change in copy number involves change in chromosome structure which eventually ends up joining two formerly separated DNA sequences, and is often mediated by DNA rearrangements. Genomic rearrangements involving gain, loss or disruption of dosage sensitive gene influence the phenotype of an organism through duplication, deletion, position effects and gene fusion or conversion events and may lead to genomic disorders [1, 3]. Since DNA rearrangements define DNA changes involving thousand to million base pairs, their formation mechanisms were speculated to be different from monogenic point mutations which are usually formed as a consequence of DNA replication errors and repair but recent evidence suggests that some disorders may be caused due to errors of replication and repair particularly involving homologous DNA strands[1-2]. Chromosome rearrangement breakpoints are scattered throughout the genome, but are more common in areas where the genomic architecture contains different repeat structures [1-2]. Accordingly, Human genome rearrangements may be divided into two major groups:

Recurring Rearrangements

These refer to rearrangements that have fixed break points i.e. they are of the same interval in different individuals.

Non Recurring Rearrangements

These refer to rearrangements having distinct breakpoints which share the smallest region of overlap (SRO) which comprises of the associated locus and its disorder.

Four mechanisms have been proposed for genomic rearrangements in the human genome:

1. Homologous Recombination

2. Non Homologous End- Joining

3. Fork Stalling and Template Switching

4. L1 Retro transposition

Figure1: Experimental observations of recurrent and non-recurrent genomic rearrangements associated with genomic disorders.

Homologous Recombination:

Homologous recombination is a characteristic feature of meiosis in diploid organisms whereby crossing over enhances the genetic diversity through exchange of genetic material and is critical for the correct alignment of chromosomes during metaphase. During the S phase of mitosis, homologous recombination between identical sister chromatids may be used to repair double strand breaks therefore rearrangements caused by NAHR is not an exclusive feature of germ cells [7-8]. Most often NAHR is responsible for recurrent genomic rearrangements and occurs between two low copy repeats or segmental duplications [4,5]. LCRs correspond to region specific DNA blocks usually of 10 to 300 kilo base in size and of > 95% to 97% similarity to each other [5,6]. Due to high degree of sequence identity, non- allelic copies of LCR, instead of the usual allelic copies get aligned together in mitosis or meiosis and cause misalignment and a subsequent crossing over between them could result in genomic rearrangements. Therefore, non allelic copies are often called substrates of Homologous Recombination. Non Allelic Homologous Recombination (NAHR) mediated by segmental duplications, accounts for most recurrent rearrangements. Segmental duplications do not mediate but stimulate non-recurrent events. However, some rare NAHR mediated by highly homologous repetitive sequences (Alu, LINE) are responsible for some non recurrent rearrangements. Nevertheless, regardless of the recombination mechanism, genomic architecture have been associated with many rearrangement breakpoints which suggests that rearrangements are not random events and may create instability in the genome.

Models of Homologous Recombination include:

Double Holliday Junction Pathway

Strand Dependent Synthesis Annealing

One Ended DSB Repair

Double Holliday Junction Pathway and Strand Dependent Synthesis Annealing:

Two ended Strand Break Repair can result either in Double Holliday Junction or in Strand Dependent Synthesis Annealing. First, double stranded breaks are created in the DNA due to exposure of a mutagen like UV, the 5' ends of DSB are then resected from 3' overhanging tails. 5' ends gets eaten away because they cannot retemplate back as replication only occurs from 3' to 5'. Coating with Rad 51 in eukaryotes then catalyses the invasion by one or both 3' ends into homologous sequence forming a D Loop. The D loop primes with the chromatid and DNA synthesis occurs. Depending on the resolution through endonucleases and ligases, this would eventually lead to a non cross- over or a cross over being produced. Resolution can be vertical or horizontal but for producing recombinants for Two ended DSB Repair, one end has to be resolved vertically and the other horizontally so that a switch over can occur. If both cuts are resolved horizontally (or vertically) then non-recombinants are produced [10].

Synthesis Dependent Strand Annealing:

SDSA is just a slight modification of Double Holliday Junction pathway and begins in the same way as the double Holliday junction pathway but differs after the polymerase extending step. In this case, Helicase first separates the double helical DNA formed of the invading and synthesized strand. The invading strand then encounters the second end from DSB and anneals through complementary base pairing. The second end is extended by DNA synthesis and is ligated later after completion[12].

Break Induced Replication Pathway:

Helicase is an enzyme responsible for converting ds DNA into ss DNA, but when it encounters a nick in the template strand, collapsed replication forks occur which begins the BIR pathway. This is a modification of SDSA, in the sense that both have invasion from 3' end but in this case particularly extension of both, leading and lagging strand occurs. However unlike in SDSA, the separated 3'end fails to find a complementary second end to anneal. This 3' end again reinvades and is extended by the low processivity of the replication fork. This trend is followed until a more processive replication fork is formed [9, 11].

During crossing over and non crossing over as well, it is evident that there would be patches of gene conversion events observed.

Unequal crossing over may result in deletion (DiGeorge Syndrome), duplication (dup22q11) or inversion (Haemophilia). Sometimes mutation from a non functional pseudo gene is inserted into the functional gene through gene conversion.

Figure 2: Mechanisms of Homologous recombination.

Figure 3: The Orientation of different segmental duplications determines the fate and type of aberration. Recombination between direct repeats results in deletion and Duplication. Recombination between inverted repeats results in inversion. Interchromosomal and interchromatid NAHR between LCRs in direct orientation results in reciprocal duplication and deletion whereas Intrachromatid NAHR only creates deletion.

Figure 4: Examples of Disorders successfully uncovered by NAHR mechanism of Structural DNA exchange include Deletion at 22q11, inversion leading to haemophilia A and Gene conversion in Congenital adrenal hyperplasia.

Although the role of NAHR as a mechanism behind structural DNA exchange has been uncovered, recent research focuses on differences in recombination frequency and homology length requirement between males and females and also between meiosis and mitosis [13-16].

Non Homologous End-joining (NHEJ):

LCR mediated NAHR does not explain all cases of genomic rearrangements. This molecular mechanism represents non recurrent chromosomal rearrangements with scattered break points. Non-homologous end joining is used to repair somatic double stranded DNA breaks primarily during G0, G1 and early S phase. It involves joining of the broken DNA strands without the use of a homologous template and utilizes very short homologous sequences (Micro homologies) to guide the repair. These micro homologies are often present as single stranded overhangs in the ends of double stranded breaks. If they are not originally present in the breakpoint then overhangs are created by removal of a few bases, which allows complementary base pairing to occur. Hence, the hallmark of this mechanism is the deletion or insertion of a few bases around the breakpoint. Ku proteins are often utilised so as to bind to free DNA ends and promote the alignment of the two DNA ends along with recruiting enzymes. Kinases are often used for processing of ends and ligase for final ligation[17-18].

Figure 5: DSB are produced due to disruption of the phosphodiester backbone of the double helix. After the detection of DSB, ku proteins align the DNA ends and protects them from degradation. This protein often engages DNA protein kinases to make the ends ligatable. artemis solely possesses 5' exonuclease activity.Artemis and DNA Pkcs possesses 5' and 3' overhang endonuclase cleavage activilty which means that this enzyme can trim 5' overhangs with a strong preference for the site that blunts the end and incontrast 3' overhangs are trimmed with a preference to leave a 4 or 5 nucleotide single stranded overhang.

Fork Stalling and Template Switching:

This mechanism represents replicative non-Homologous repair system of DNA exchange. With the advent of more sophisticated scientific technology, complex details of genomic rearrangements can be observed which has led to the proposal of DNA replication based FoSTeS model as a potential mechanism for DNA Exchanges. Certain diseases like PMD (Pelizaeus- Merzbacher Disease) could not be fully explained on the basis of NAHR and NHEJ mechanism [19].

Accordingly, during replication when replication fork stalls at one position, the lagging strand disengages from the template and anneals via 3' end homology to another replication fork which is in the nearest vicinity and restarts the DNA synthesis. Invasion and annealing are dependent upon the microhomology between invaded and original site. Switching to another fork located downstream (forward invasion) would result in deletion whereas switching to fork located upstream (backward invasion) results in duplication and depending on whether lagging or leading strand in the new fork was invaded and copied along with the direction of fork progression, the erroneously incorporated fragment from the new replication fork would be in direct or inverted orientation to its original position and this invading step could be repeated multiple times which would ultimately reflect low processivity of polymerase[19].

Figure 6: Genomic rearrangement mechanism: 1. After the original stalling of the replication fork, the lagging strand disengages and anneals to a second fork via micro homology. 2. Extension of primed second fork and DNA synthesis. After fork disengages 3. The tethered original fork with its lagging strand may invade a third fork and this could occur several times before 4. Resumption of replication on the original template.

L1 Retrotransposition:

These represent the human genetic elements which insert their extra copies throughout the genome through cut and paste mechanism. LI Elements constitute almost ~20% of mammalian genomic DNA content. Most of these are retrotransposition incompetent because of truncated L1 copies but 150 full length L1 elements are present within the human genome [20-23].

Structure:

Full length Non-LTR autonomous L1 is about ~6kb long and consists of a 5' UTR region containing an internal RNA polymerase II (RNAP II) promoter [24], two open reading frames (ORF 1 and ORF 2) and a 3' UTR containing poly A tail signal. ORF 1 encodes for an RNA binding protein and ORF 2 codes for protein with endonuclease and reverse transcriptase function [25]. This constitutes the equipment for Target primed Reverse Transcription therefore making L1 elements the sole autonomous transposon elements in the human genome.

Because of TPRT and decay over time, most of the L1 copies become inactivated by truncations, internal rearrangements and mutations [26-27]. There is evidence of more than 500000 L1 copies in the human genome, out of which less than 100 happen to be functional [28].

The Retrotransposition Cycle:

RNA polymerase II mediates the transcription of L1 locus through an internal promoter which in turn directs transcription initiation at the 5' end of L1 transposon [29]. Because of this internal promoter that RT is able to generate autonomous duplicate copies at different locations in the genome.

The transcript is then transferred to the cytoplasm where ORF 1 and ORF 2 are translated. Since both proteins produced show a cis preference [30] therefore they associate with the transcript that encoded them to produce a ribonucleoprotein (RNP) particle. This RNP is then transported back into the nucleus by a mechanism which is not understood properly uptil now.

Figure 7: The Retro transposition Cycle.

The integration of L1 element into the genome is thought to occur through "Target - primed Reverse Transcription" (TPRT) [31-33].

During TPRT, L1 endonuclease cuts the first strand of target DNA, generally between T and A at 5'TTTTAA3' consensus sites [34]. Consequently, the free 3'OH liberated as an aftermath of the cut at the first strand, is used to prime reverse transcription of L1 RNA by L1 Reverse Transcriptase. After cutting the second strand of target DNA, it is used to prime second strand synthesis producing Target site duplications.

Figure 8: Target primed reverse transcription.

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