Objectives of DNA recombination

• The different processes of DNA recombination: homologous recombination, site-specific recombination, transposition, illegitimate recombination, etc.

• What are the differences between these process: (i) the DNA substrates, (ii) the enzymes used, and (iii) the recombinant products produced.

• General mechanism of recombination: (I) presynapsis (initiation), (ii) synapsis (the formation of joint molecules), and (iii) postsynapsis (resolution).

• In addition to provide genetic diversity, DNA recombination plays an important role in repair of DNA double-strand breaks and DSG (to be discussed in the section of DNA repair).

Examples of recombination

Homologous recombination• Refer to recombination between homologous DN

A sequence in the same or different DNA molecules.

• The enzymes involved in this process can catalyze recombination between any pair of homologous sequences, as long as the size of homologous sequence is longer than 45 nt or longer. No particular sequence is required.

• Models of homologous recombination.• Homologous recombination of E. coli.• Meiotic recombination.

The Holliday model of recombination

Homologous recombination of E. coli

• Identification of genes involved in recombination: (i) isolation of mutants affecting recombination in wild-type cells (eg., recA, recB, recC etc.), (ii) the recombinational deficiency in recBC cells may be suppressed by sbcA or sbcB mutations. The sbcB gene encodes for a 3’ to 5’ ss-DNA exonuclease, while the sbcA mutation activate the expression of recE which encodes for 5’ to 3’ exonuclease. (iii) isolation of mutants affecting recombination in recB recC sbcB or recB recC sbcA cells (eg., recF, recO, recR, recQ, recJ etc.)

• The biochemical functions of rec genes.

Homologous recombination is catalyzed by enzymes

• The most well characterized recombination enzymes are derived from studies with E. coli cells.

• Presynapsis: helicase and/or nuclease to generate single-strand DNA with 3’-OH end (RecBCD) which may be coated by RecA and Ssb.

• Synapsis: joint molecule formation to generate Holliday juncture (RecA).

• Postsynapsis: branch migration and resolution of Holliday juncture (RuvABC).

RecBCD

• A multifunctional protein that consists of three polypeptides RecB (133 kDa), RecC (129 kDa) and RecD (67 kDa).

• Contain nuclease (exonuclease and Chi-specific endonuclease) and helicase activity.

5‘-GCTGGTGG-3’

Chi-specific nicking by RecBCD

Fig. 22.7

Helicase and nuclease activities of the RecBCD

The Bacterial RecBCD System Is Stimulated by chi Sequences

FIGURE 15.17: RecBCD unwinding and cleavage

The RecBCD pathway of recombination

RecA binds selectively to single-stranded DNA

Fig. 22.4

RecA forms nucleoprotein filament on single-strand DNA

Fig. 22.5

Paranemic joining of two DNA (in contrast to plectonemic)

Fig. 22.6

Strand-Transfer Proteins Catalyze Single-Strand Assimilation

• RecA forms filaments with single-stranded DNA and catalyzes the assimilation of single-stranded DNA to displace its counterpart in a DNA duplex.

FIGURE 15.18: RecA strand invasi

on

RuvABC

• RuvA (22 kDa) binds a Holliday junction with high affinity, and together with RuvB (37 kDa) promotes ATP-dependent branch migration of the junctions leading to the formation of heteroduplex DNA.

• RuvC (19 kDa) resolves Holliday juncture into recombinant products.

Fig. 22.9

Fig. 22.10

Fig. 22.14

Fig. 22.15

Fig. 22.17

Homologous Recombination Occurs between Synapsed Chromosomes in Meiosis

• Chromosomes must synapse (pair) in order for chiasmata to form where crossing-over occurs.

• The stages of meiosis can be correlated with the molecular events at the DNA level.

FIGURE 03: Recombination

occurs at specific stages

of meiosis

Fig. 15.13

Fig. 15.15

The Synaptonemal Complex Forms after Double-Strand Breaks

• Double-strand breaks that initiate recombination occur before the synaptonemal complex forms.

• If recombination is blocked, the synaptonemal complex cannot form.

• Meiotic recombination involves two phases: one that results in gene conversion without crossover, and one that results in crossover products.

Fig. 22.18

Fig. 22.19

Fig. 22.20

Fig. 22.21

Fig. 22.24

Gene conversion: the phenomenon that abnormal ratios of a pair of parental alleles is detected in the meiotic products. At the molecular level: the conversion of one gene’s sequence to that of another.

Fig. 22.25

Fig. 22.26

Site-specific Recombination: Bacteriophage lambda integration in E. coli

Fig. 15.28

A site-specific recombination reaction (eg. catalyzed by Int of bacteriophage lambda)

Fig. 15.31

Recombination Pathways Adapted for Experimental Systems

FIGURE 15.38: Cre/lox system for gene kno

ckouts

Adapted from H. Lodish, et al. Molecular Cell Biology, Fifth edition. W. H. Freeman & Company, 2003.

Fig. 23.21

Fig. 23.12

Fig. 23.13

Fig. 23.14

Fig. 23.15

Fig. 23.16

Fig. 23.17

Transposition• Transposition is mediated by transposable elements, or

transposons.• Transposons of bacteria: IS (insertion sequences) cont

ains only sequences required for transposition and proteins (transposases) that promote the process. Complex transposons contain genes in addition to those needed for transposition.

• Transposition is characterized by duplication of direct repeats (5-9 bps in most cases) at target site.

• Transposition, in some instances, may be mediated through a RNA intermediate (retrotransposons and non-LTR retrotransposons).

Duplication of the DNA sequence at a target site when a transposon is inserted

Fig. 23.1

Fig. 23.2

Fig. 17.3

Fig. 23.3

Fig. 23.4

Fig. 23.5

Replicative transposition is meidatedby a cointegrate intermediate.

Fig. 23.6

Fig. 23.7

Eukaryotic transposons

• DNA transposons: (i) Ds and Ac of maize, (ii) Drosophila P elements.

• Retrotransposons: (i) LTR retrotransposons (Ty element of yeast and copia of Drosophila). (ii) non-LTR retrotransposons (LINES, Alu, group II introns).

Ds and Ac of maize

Fig. 23.8

Fig. 23.9

Fig. 23.10

Hybrid Dysgenesis

FFig. 17.20

Fig. 17.21

Fig. 17.22

Fig.23.19

Fig. 23.18

Fig. 23.20

Fig. 23.21

Fig. 23.22

Fig. 23.23

Fig. 23.24

Nonviral transposons: LINES

Fig. 23.25

Fig. 23.26

Fig. 23.27

Fig. 23.28

Group II introns: Retrohoming

DNA Repair

• DNA damage may arise: (i) spontaneously, (ii) environmental exposure to mutagens, or (iii) cellular metabolism.

• DNA damage may be classified as: (I) strand breaks, (ii) base loss (AP site), (iii) base damages, (iv) adducts, (v) cross-links, (vi) sugar damages, (vii) DNA-protein cross links.

• DNA damage, if not repaired, may affect replication and transcription, leading to mutation or cell death.

Fig. 20.27

Fig. 20.28

Fig. 20.29

Methylataion and Mismatch Repair

Model for Mismatch Repair

Base-Excision Repair

FIGURE 16.12: Uracil is removed from DNA

FIGURE 16.13: Glycosylases remove bases

16.5 Base Excision Repair Systems Require

Glycosylases

FIGURE 16.14: Base removal triggers excision repair

Nucleotide-Excision Repair in E. coli and Humans

Alkylation of DNA by alkylating agents

Direct Repair: Photoreactivation by photolyase

O6-methyl G, if not repaired, may produce a mutation

Direct Repair: Reversal of O6 methyl G to G by methyltransferase

Direct re

Direct repair of alkylated bases by AlkB.

Effect of DNA damage on replication: (i) coding lesions won’t interfere with replication but may produce mutation, (ii) non-coding lesions will interfere with replication and may lead to formation of daughter-strand gaps (DSG) or double-strand breaks (DSB).

DSG and DSB may be repaired by recombination process, to be discussed in the following section.

Models for recombinational DNA repair

Fig. 20.40

Fig. 20.38

Model for nonhomologous end-joining

Figure 16.25: NHEJ requires several reactions.

Fig. 20.41