Molecular Biology Chapter 10

Front 1

Key steps of homologous recombination shared by models

Back 1

1. alignment of two homologous DNA molecules
2. introduction of breaks in the DNA.
3. formation of initial short regions of base pairing between the two recombining DNA molecules. strand invasion, in which a ss region of one molecule base pairs with the other molecule.
4.movement of the holliday junctions by the repeated melting and formation of bps. branch migration, promoting further exchange of DNA.
5.Resolution, cleavage of hte Holliday junction separates the two molecules.

Front 2

heteroduplex

Back 2

Bb in the middle after branch migration.

Front 3

Holliday junction cleavage

Back 3

two alternative pairs of DNA sites can be cut during resolution. one splice and one patch

Front 4

patch

Back 4

non-crossover products. strands that were broken to initiate recombination

Front 5

splice

Back 5

crossover products. DNA strands that were not broken during initiation rxn.

Front 6

DSB repair pathway

Back 6

initiating event is the introduction of a DSB in one of the two DNA molecules. a DNA cleaving enzyme sequentially degrades the broken DNA molecule to generate regions of ssDNA, creating ssDNA tails terminate with 3' ends. they then invade the unbroken homologous DNA duplex forming a D-loop. 3' ends can serve as primers.

Front 7

how double strand breaks form

Back 7

damage in the DNA template can lead to DSM formation during DNA replication. Nick in strand or Lesion.

Front 8

RecBCD pathway

Back 8

proteins that promote recombination in E/ coli via this major DSB repair pathwya. requires a DSB on one of the recombining two DNA molecules. uses ATP-hydrolysis. has two helicases and an endonuclease activity that generates a 3' overhang. it unwindes and degrades both DNA strands until a chi sequence is reached at which the 3' end is protected. mechanism destroys invading DNA while promotes exchange with E.coli DNA.

Front 9

chi

Back 9

crossover hotspot instigator. stimulates the frequency of homologu recombination.
GCTGGTGG

Front 10

RecA

Back 10

promotes strand exchange. Like SSB, it binds to ssDNA forms fimlament around DNA. needs at least 15 bp. has room to associate double stranded template. Filament has two binding sites: 1. primary bound by the first DNA molecule. 2. seconday binding is rapid weak transient and independent of DNA sequence.

Front 11

Polarity of RecA

Back 11

new subunits of RecA join the filament on the DNA 3' side to an existing subunite much faster than those on the 5' side. DNA molecules with 3' ssDNA extensions iwll be efficiently coated with Reca. those with 5' ssDNA would not serve as substrates for filament assembly.

Front 12

RuvAB complex

Back 12

promotes holliday junction migration. RuvA recognizes Holliday junctions and recruites RuvB helicase. RuvB uses ATP to wind DNA through each RuvB, causing migration of junction. RuvA is a tetramer. RuvB is a hexamer.

Front 13

RuvC

Back 13

resolves holliday junctions by cleaving across the junction separating the two DNA molecules.

Front 14

non-disjunction

Back 14

the failure to segregate homologous chromosomes to opposite progeny cells. the result of failure of rercombination.

Front 15

what is the result of no recombination?

Back 15

chromosomes often fail to aligh properly for the first meiotic division and as a result there is a high incidence of chromosome loss. often reflected in poor fertility.

Front 16

Spo11

Back 16

this gene encodes a protein that introduces dsbs in chromosomal DNA with a mechanism similar to topoisomerase to initiate meiotic recombination. Cuts at many different locations. the OH group of a tyrosine in the Spo11 protein attacks the DNA to form a covalent protein - DNA linkage. Two subunits of Spo22 are required to generate a double-stranded DNA break, one to attack each of he two DNA strands. Because of this cleavage mechanism, the DSB can be resealed by the simple reversal of the cleavage reaction.

Front 17

Meiotic recombination pathway

Back 17

both Spo11 and MRX complex need to be present to form DSBs during meiosis. MRX protein is responsible for resection of the 5'-ending strands at the break site. the strand exchange proteins Dmc1 and Rad51 then assemble on the ssDNA tails. both proteins participate in recombination, but how they work together is not known.

Front 18

complementation

Back 18

in diploid cells, mutations usually do not cause immediately observable phenotypes because the second functional copy of the gene compensates for the lack of function of the mutant allel.

Front 19

eseentail genes

Back 19

genes whose deletion (null mutation) causes lethality of the organism

Front 20

hartwell

Back 20

screened for temperature sensitive strains.