Genetic Recombination DNA Recombination - VCU eCurriculum [PDF]

5. Prokaryotic: During the following processes, homologous recombination takes place. a. Conjugation (mating) – When a

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Idea Transcript


M1 – Biochemistry

Genetic Recombination Dr. Kordula

1

DNA Recombination

1.

Homologous genetic recombination (general recombination)

2. 3.

Site-specific recombination DNA transposition

2

General Recombination A. Exchange of sections of homologous, nonnon-identical DNA molecules. Eukaryotic: During the process of meiosis, diploid cells undergo one DNA replication and two cell divisions giving rise to haploid gametes. During this process, homologous chromosomes pair; i.e., chromosomes that encode for the same gene products and which are similar in DNA sequence, form a complex that allows homologous sequences to contact each other. A small percent of mismatch between homologues strands is tolerated. The general recombination mechanism allows portions of mom's DNA to be exchanged with the similar portions of dad's DNA resulting in greater shuffling of the genes in the gene pool. This increased diversity of gene combinations of the mating population enhances the chance of survival in a changing environment.

3

Meiotic recombination

Recombination

4

Prokaryotic: During the following processes, homologous recombination takes place. a. Conjugation (mating) – When a portion of the chromosome of a donor cell is transferred to a recipient cell, homologous sequences promote crossing over. b. Transducing phage – When bacterial DNA is "accidentally" packaged into a phage particle and introduced into a different host cell via the normal infection process, homologous sequences can recombine. c. Transformation – When naked DNA from the environment is taken up by a bacterium, homologous sequences will promote recombination.

5

Characteristics of the general recombination 1.

Homologous sequences are exchanged.

2.

The process occurs wherever homologous sequences exist.

3.

Resulting joints are staggered.

4.

No nucleotides are ultimately gained or lost at the exchange site, yet some mismatch is tolerated. 6

Basis of the mechanism lies in the denaturation - renaturation process Denaturation provides single-stranded regions on one duplex that seek out like sequences on another duplex. Renaturation of complementary single-strand regions of each homologue brings the two interacting duplexes together in register.

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Most of early information on the mechanism has been obtained from the study of RecBCD and RecA protein in bacteria. 1. RecBCD - multifunctional a. helicase activity (ATP required) b. ATP-dependent single-strand exonuclease activities 5’ to 3’ and 3’ to 5.’ At a Chi sequence, an octamer (GCTGGTGG) that occurs every 10 kb in the E. coli chromosome, the 3’ exonuclease is slowed and the 5’ exonuclease is enhanced, giving rise to a 3’ overhang whisker. 5’ 3’

5’

Chi 3’

RecBCD

5’ 3’

3’ 5’

Note: Fear not! The erased base pairs in the gap will be replaced replaced later under the direction of the second duplex as the template. 8

2. RecA – multifunctional a. binds ssDNA cooperatively and directionally. b. RecA-ssDNA complex binds to dsDNA (ATP required). c. enhances process of branch migration (ATP + Pi).

ADP

9

Specifics of General (Euk. - Meiotic) Recombination

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Specifics of General Recombination

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Specifics of General Recombination

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Specifics of General Recombination

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Specifics of General Recombination

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Specifics of General Recombination

Patch

15 Splice

Meselson-Radding Model Branch migration The origin of the double-strand break in the original duplex remains mysterium tremendum. Let’s just say it happens. (This problem is solved by Meselson-Radding’s single nick and strand invasion model). The process of branch migration that elongates the region of interaction between the two duplexes is worthy of further emphasis and clarification. Note that branch migration is promoted by RecA binding and results in the breaking of old complementary pairs and the making new complementary pairs, as diagramed in the triplex structure below.

Note: The degree of branch migration dictates the length of the heteroduplex, cross-over junction. 16

The Holliday Junction

The Holliday Junction (Crossstrand exchange). This structure forms when two cleavages and two ligations covalently lock the interacting duplexes to each. 17

Branch Migration, Holliday Junction

Branch Migration

18

The Resolution of the Holliday junction The isomerization of the Holliday junction and two more cleavages plus two ligations completes the recombination yielding either the spliced recombinant (complete crossing-over) or the patch recombinant (single strand exchange).

Splice

Patch

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Combining Repair and Recombination to Correct Double-Strand Breaks Accidental double-strand breaks in DNA can be dealt with using both processes. This mechanism has particular relevance in human disease in that two of the involved proteins are BRCA-1 and BRCA-2. It is known that a large percentage of women who inherit a mutation in one allele of either BRCA-1 or BRCA-2 are predisposed to either breast cancer or ovarian epithelial cancer. The general process of repair of doublestrand breaks by homologous recombination is diagramed below.

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Combining Repair and Recombination to Correct Double-Strand Breaks (cont.)

21

Combining Repair and Recombination to Correct Double-Strand Breaks (cont.)

22

Combining Repair and Recombination to Correct Double-Strand Breaks (cont.)

23

Site-Specific Recombination In contrast to homologous recombination which permits interaction of any homologues, the site-specific mechanism occurs only at specific sequences. The general term for the enzyme involved is recombinase. These enzymes catalyze the integration of one DNA into another DNA. Examples include lambda DNA integration and transposon integration. Those specific mechanisms are shown below. Note that the lambda recombinase is called integrase and the transposon recombinase is called transposase.

Cre recombinase

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λ phage integration/excision

25

Transposable elements Transposable elements are DNA sequences that do not have a fixed position in a genome, but rather can move from place to place within the genome. These jumping genes have been demonstrated in prokaryotes and eukaryotes. The process requires little, or no, sequence homology and the element itself encodes for the transposase enzyme that carries out the process.

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Duplication of the DNA sequence at a site of integration

27

Transposition can cause the following sequelae: 1. The process involves duplication of the target site (3-12 bp) 2. Some DNA synthesis may be involved. 3. The element itself may be duplicated. 4. The process may restructure the chromosome by causing deletions or insertions, leading to gene inactivation. 5. A gene may be activated due to moving a promoter into juxtaposition. 6. Some transposons carry and confer antibiotic resistance.

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Eukaryotic Transposons Classes of the DNA sequences found in the human genome Protein-encoding genes: 1% Transcribed exons, 26,000 (40,000 genes?) (~ 30-40 Mbp) Introns: 24% Non-coding DNA comprising; majority of most genes Structural DNA: 20% Constitutive heterochromatin, localized near centromeres and telomeres Repeated sequences: 3% Simple sequence repeats (SSRs) of a few nucleotides repeated millions of times

29 and…

Eukaryotic Transposons Transposable elements: 45% 20% long interspersed elements (LINEs); potentially active retrotransposons (~ 6 kb; encode RT) 10% “parasitic” ALU sequences; ~ 1.2 X 106 copies ~300 bp long, uses LINE’s machinery • associated with some harmful mutations 15% other transposable elements 8% retrotransposons 3% DNA transposons 4% “dead” transposons Estimate: - Alu transposition event in humans, 1/200 newborns - may have occurred 1 per live birth millions of years ago - more active elements more likely to kill hosts.

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