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Score Distribution

0

24

6

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1012

14

16

1820 Average: 70.3

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Chapter 15

Homologous and Site-Specific

Recombination

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15.1 Introduction

Three types of recombination Homologous recombination Site-specific recombination Somatic recombination (e.g., immune cells)

Homologous recombination is essential in meiosis forcreating genetic diversity and for chromosome

segregation, and in mitosis to repair DNA damage andstalled replication forks.

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15.1 Introduction

Site-specific recombination involves specific DNAsequences. somatic recombination Recombination that occurs in

nongerm cells (i.e., it does not occur during meiosis);

most commonly used to refer to recombination in theimmune system.

Figure 15.03: Site-specific recombination

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15.2 Homologous Recombination Occurs between

Synapsed Chromosomes in Meiosis

Sister chromatids

Sister chromatids

bivalent = 4 DNA duplexes

chiasma (pl. chiasmata)

Figure 15.02

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Sister chromatid: each of two identical copies of areplicated chromosome; this term is used for the two

copies linked at the centromere

Bivalent: the structure containing all four chromatids atthe start of meiosis

Chiasma: site at which two homologous chromosomeshave exchanged material during meiosis.

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Figure 15.05: Recombination involves pairing between complementary strands ofthe two parental DNAs.

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Recombination is initiated by makingadouble-strand break (DSB)in one(recipient) DNA duplex.

Exonuclease action generates 3!single-stranded ends (5 end

resection).

Free 3 ends invade the other (donor)duplex single strand invasion.

When a single strand from recipientdisplaces its counterpart in donor, it

creates a D-loop, which grows as

DNA synthesis occurs. This generates a recombinantjoint

molecule in which the two DNA

duplexes are connected by

heteroduplex DNA.

15.3 Double-Strand Breaks Initiate Recombination

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branch migration The ability of a DNAstrand partially paired with its complement ina duplex to extend its pairing by displacingthe resident strand with which it ishomologous.

Resolutionrequires a further pair of nicks.

Resolution occurs at Holiday junction. Whether recombinants are formed dependson whether the strands involved in theoriginal exchange (same strands)or theother pair of strands (other strands)arenicked during resolution. movie

Splice recombinants vs. patch recombinants A strand exchange between duplex DNAs

always leaves behind a region of heteroduplex DNA, but it may or may not be

accompanied by recombination of theflanking regions.

15.3 Double-Strand BreaksInitiate Recombination

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15.4 Recombining Chromosomes Are Connected

by the Synaptonemal Complex

During the earlypart of meiosis,

homologous

chromosomes are

paired in the

synaptonemal

complex. The mass of

chromatin of each

homolog is

separated from

the other by a

proteinaceouscomplex.

Figure 15.10

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15.4 Recombining Chromosomes Are Connected

by the Synaptonemal Complex

Cohesins and Zip proteinsform the lateral elements andtransverse filaments/central elements.

axial element A proteinaceous structure around whichthe chromosomes condense at the start of synapsis.

lateral element A structure in the synaptonemalcomplex that forms when a pair of sister chromatids

condenses on to an axial element.

central element A structure that lies in the middle ofthe synaptonemal complex, along which the lateral

elements of homologous chromosomes align.

It is formed from Zip proteins.

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15.5 Specialized Enzymes Catalyze 5"EndResection and Single-Strand Invasion

DSB is generated during meiosis by Spo11 (see Fig. 15.7) MRN (or MRX in yeast) complexes:

are required for Spo11 displacement are required for 5"end resection (CtIP endonuclease and

Exo I are also needed) prevent separation of broken DNA ends. MRN = Mre11, Rad50, Nbs1 MRX = Mre11, Rad50, Xrs2

Single strand invasion: RecA(Rad51/Dmc1in eukaryotes)-type proteins form presynaptic filamentswith single-stranded or duplex DNA and catalyze the ability of a single-

stranded DNAwith a free 3!to displace its counterpart in a

DNA duplex.

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15.6 Holliday Junctions Must Be Resolved

RuvArecognizes the structure of the junction and RuvBis a helicase that catalyzes branch migration!branchmigration determines the length of the regions of

heteroduplex DNA.

Figure

15.14

E. Coli system

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15.6 Holliday JunctionsMust Be Resolved

RuvC(endonuclease) cleavesjunctions to resolve Holiday

junctions.

RuvC preferentially cleavesATTG, which may direct which

strand is cleaved.

Resolvases (junction-resolving enzymes) sharemoderate structural similarity

!cleavage mechanism may

be diverse.

Figure 15.15: Complex of T4 endo VII, with the two subunits colored differently(blue and green) bound to a Holliday junction (each DNA strand is color-coded.)

E. Coli system

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15.8 Site-Specific Recombination ResemblesTopoisomerase Activity

Site-specific recombination involves a reaction betweenspecific sites that are not necessarily homologous. The length of target sites are short (14~50 bp). recombinase enzyme that catalyzes site-specific

recombination; more than 100 recombinases are known. Examples of integrase family (belongs to recombinase): Int

from phage lambda, Crefrom phage P1, FLPfrom yeast.

" Target sites for integrases- Int (integrase): att(attachement)- Cre (causes recombination): loxP[locus of crossing (x) over, P1)- Flp (flippase): FRT(flipase recognition target)

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15.8 Site-Specific Recombination ResemblesTopoisomerase Activity

Int-attrequires differentfactors for integration andexcision. Target sites are

different.

Cre-loxP: Cre is sufficientfor both integration andexcision (no accessary

proteins). Target sites are

identical.

Flp-FRT: target sites areidentical.

Figure 15.18

O, core sequenceB, B, P, P: arms

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15.8 Site-Specific Recombination Resembles

Topoisomerase Activity

DNA phosphodiester bond is broken by nucleophilic attack bytyrosine (Tyr) or serine (Ser) on recombinases.

A phosphodiester bond is formed between recombinase andDNA andfree 5-OHend is released.

The free 5-OH attacks 3-phosphoTyr line in the other siteand link the two broken ends.

No additional energy is needed = energy is conserved.

Figure 15.19

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15.8 Site-Specific Recombination Resembles

Topoisomerase Activity

Figure 15.19

Holiday junctio

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15.9 Yeast Use a Specialized RecombinationMechanism to Switch Mating Type

Figure 15.21: The yeast life cycle

a

factor

!

factor

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The yeast mating typelocus MAT, a mating-type cassette, has either

the MATaor MAT#

genotype.

The allele at MATiscalled the active cassette.

There are also two silentcassettes, HML#and

HMRa. They are in

heterochromatin.

Figure 15.22

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MAT, HML#

and HMRaarehomologous.

Yregion is similar andflanking regions haveidentical sequence.

Y region only in activecassettecan be cleaved by

HO endonuclease, whichinitiate a speicial

homologous recombinationwith a different silentcassette (80~90% of time).

!gene conversion

Figure 15.23

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Figure 15.25

HO generates site-specific DSBs (just right of the Y boundary).

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Figure B15.1

Trypanosomes use gene switching to evade the host immune system.

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Trypanosomes use gene switching to evade the host

immune system.

More than 20 various surface glycoprotein(VSG) genes arepresent; however, only one gene is expressed at any given time.

Only one VSGgene near telomere is active and the others aresilenced.

An inactive VSGis recombined with the active VSGand changecoat!immune evasion.

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