chapter10 homologous recombination at the molecular level 200431760044 顾辉辉...

CHAPTER10

Homologous Recombination at the Molecular Level

200431760044 顾辉辉生物科学第四组

Outline Models for homologous recombinationHomologous recombination protein

machinesHomologous recombination in eukaryotesMating-type switchingGenetic consequences of the mechanism

of homologous recombination

Models for homologous recombination

Key steps of homologous recombination shared by different models

Alignment of two homologous DNA molecules Introduction of breaks in the DNA Formation of initial short regions of base

pairing between the two recombining DNA molecules

Movement of the Holliday junction Cleavage of the Holliday junction

The Holliday model illustrates key steps in homologous recombination

The Holliday model very well illustrates the DNA strand invasion, branch migration, and ,Holliday junction resolution processes central to homologous recombination

Holliday model through the steps of branch migration

Holliday junction cleavage

Double –stranded DNA breaks arise by numerous means and initiate homologous recombination

Double-straned breaks in DNA arise quite frequently

In bacteria, the major biological role of homologous recombination is to repair DSBs

In addition to repairing DSBs in chromosomal DNA, homologous recombination promotes genetic exchange in bacteria

In eukaryotic cells, homologous recombination is critical for repairing DNA

Damage in the DNA template canlead To DSB formation during DNA replication

Homologous recombination protein machines

The recBCD helicase nuclease processes broken DNA molecules for recombination

RecA protein assembles on single-stranded DNA and promotes strand invasion

Newly base-paired partners are eatablidshed within the recA filament

RuvAB complex specifically recognizes Holliday junctions and promotes branch migration

RuvC cleaves specific DNA strands at the Holliday junction to finish recombination

The recBCD helicase nuclease processes broken DNA molecules for recombination DNA molecules with single-stranded DNA ext

ensions or tails are the preferred substrate for initiating strand exchange between regions of homologous sequence

The RecBCD enzyme processes broken DNA molecules to generate these regions of ssDNA

RecBCD provide a means for cells to choose

Steps of DNA processing By RecBCD

Polar action of chi

RecA protein assembles on single-stranded DNA and promotes strand invasion

RecA is the central protein in homologous recombination

RecA makes DNA pairing and strand -exchange

The active form of RecA is a protein-DNA filaments

Examples of DNA paring and strand -exchange reaction

Three view of the RecA filament

Polarity of RecA assembly

Newly base-paired partners are established within the RecA filament

RecA-catalyzed strand exchange can be divided into different stages

cross-section of single DNA strand bound to RecA protein

DNA in secondary site is testes for complementarity

Base -paring between strands is switched

Model of two steps in search for homology and DNA strand exchange within the RecA filament

RecA homologous are present in all organisms

Strand-exchange proteins of the RecA family are present in all forma of life

RecA-like protein in three branches of life

RuvAB complex specifically recognizes Holliday junctions and promotes branch migration

RuvA protein is a Holliday junction specific DNA-binging protein that recognizes the structure of the DNA junction

RuvB is a hexametric ATPase

High resolution structure of the RuvA-DNA complex and schematic model of the RuvAB complex bound to Holliday junction DNA

RuvC cleaves specific DNA strands at the Holliday junction to finish recombination

Completion of recombination requires that the Holliday junction between the two recombining DNA molecules be resolved

RuvC specifically nicks two of the homologous DNA strands that have the same polarity

RuvC cleaves DNA with modest sequence specificity

High resolution structure of the RuvC resolves and schematic model of the RuvC dimer bound to Holliday junction DNA

Homologous recombination in eukaryotes

Homologous recombination has additional functions in eukaryotes

Homologous recombination is required for chromosome segregation during meiosis

Programmed generation of double –stranded DNA breaks occurs during meiosis

MRX protein processes the cleaved DNA ends for assembly of the RecA-like strand –exchange proteins

Dmc1 is a RecA-like protein that specifically functions in meiotic recombination

Many protein function together to promote meiotic recombination

Homologous recombination has additional functions in eukaryotes

Homologous recombination can repair double –stranded breaks in DNA , to restart collapsed replication forks, and to allow a cell’s chromosomal DNA to recombine with DNA that enters via phage infection or junction

During meiosis, homologous recombination is required for proper chromosome pairing

Recombination must be complete before the first nuclear division to allow the homologs to properly align and then separate

The improper segregation of chromosomes, called nondisjunction, leads to a large number of gametes without the correct chromosome complement

The homologous recombination events that occur during meiosis are called meiotic recombination

Homologous recombination is required for chromosome segregation during meiosis

DNA dynamics during meiosis

Cytological view of crossing over

Programmed generation of double –stranded DNA breaks occurs during meiosis

The spo11protein cuts the DNA at many chromosomal locations

The mechanism of DNA cleavage The cleavage involves a covalent protein –

DNA complex has two consequences

Mechanism of cleavage by spo11

MRX protein processes the cleaved DNA ends for assembly of the RecA-like strand –exchange proteins

The DNA a5 5he site Spo11-cataluzed double-strand break is processed to generate dingle-stranded regions needed for assembly aof the RecA-like strand-exchange protein

Processing of the DNA at the break site occurs exclusively on the DNA strand that terminates with a 5’ end

Overview of meiotic recombination pathway

Dmc1 is a RecA-like protein that specifically functions in meiotic recombination

Eukaryotes encode two well-characterized homologs of the bacterial RecA protein: Rad51 and Dmc1

Dmc1 is expressed only as cells enter meiosis

strand exchange during meiosis occurs between a particular type of homologous DNA partner

Dmc1-dependent recombination occurs preferentially between nonsister homologous chromatids

Many protein function together to promote meiotic recombination

Some large protein-DNA complexes, known as recombination factories

Rad51, Dmc1, Rad52……

Co –localizations of the rad51 and Dmc1 proteins to “recombination factories” in cells undergoing meiosis

mating-type switching

Mating-type switching is initiated by a site-specific double-strand break

Mating-type switching is a gene conversion event, not associated with crossing over

Mating-type switching is initiated by a site-specific double-strand break

Mating-type switching is initiated by the introduction of a DSB at the MAT locus

This reaction is performed by a specialized DNA-cleaving enzyme, called the HO endonuclease

Mating-type switching is unidirectional

Genetic loci encoding mating-type information

Mating-type switching is a gene conversion event, not associated with crossing over

Models for recombination that do not involve Holliday junction intermediates better explain mating-type switching

Synthesis-dependent strand annealing Completing recombination requires that the

other “old” DNA strand present at MAT be removed

Recombination model for mating-type switching: synthesis-dependent strand annealing

Genetic consequences of the mechanism of homologous recombination A corollary of the fact that recombination is g

enerally independent of sequence is that the frequency of recombination between any two genesis generally proportional to the distance between those genes

A region of DNA does not have the “average” probability of participation in recombination

Gene conversion occurs because DNA is repaired duringrecombination

Comparison of the genetic and physical maps of a typical region of a yeast chromosome

Mismatch repair of heteroduplex DNA within recombination intermediates can give rise to gene conversion