...

DoubleStranded DNA Molecules with Similar Sequences Sometimes Recombine

by taratuta

on
Category: Documents
54

views

Report

Comments

Transcript

DoubleStranded DNA Molecules with Similar Sequences Sometimes Recombine
III. Synthesizing the Molecules of Life
27. DNA Replication, Recombination, and Repair
27.5. Double-Stranded DNA Molecules with Similar Sequences Sometimes
Recombine
Most processes associated with DNA replication function to copy the genetic message as faithfully as possible.
However, several biochemical processes require the recombination of genetic material between two DNA molecules. In
genetic recombination, two daughter molecules are formed by the exchange of genetic material between two parent
molecules (Figure 27.37).
1. In meiosis, the limited exchange of genetic material between paired chromosomes provides a simple mechanism for
generating genetic diversity in a population.
2. As we shall see in Chapter 33, recombination plays a crucial role in generating molecular diversity for antibodies and
some other molecules in the immune system.
3. Some viruses utilize recombination pathways to integrate their genetic material into the DNA of the host cell.
4. Recombination is used to manipulate genes in, for example, the generation of "gene knockout" mice (Section 6.3.5).
Recombination is most efficient between DNA sequences that are similar in sequence. Such processes are often referred
to as homologous recombination reactions.
27.5.1. Recombination Reactions Proceed Through Holliday Junction Intermediates
The Structural Insights module for this chapter shows how a recombinase
forms a Holliday junction from two DNA duplexes and suggests how this
intermediate is resolved to produce recombinants.
Enzymes called recombinases catalyze the exchange of genetic material that takes place in recombination. By what
pathway do these enzymes catalyze this exchange? An appealing scheme was proposed by Robin Holliday in 1964. A
key intermediate in this mechanism is a crosslike structure, known as a Holliday junction, formed by four polynucleotide
chains. Such intermediates have been characterized by a wide range of techniques including x-ray crystallography
(Figure 27.38). Note that such intermediates can form only when the nucleotide sequences of the two parental duplexes
are very similar or identical in the region of recombination because specific base pairs must form between the bases of
the two parental duplexes.
How are such intermediates formed from the parental duplexes and resolved to form products? Many details for this
process are now available, based largely on the results of studies of Cre recombinase from bacteriophage P1. This
mechanism begins with the recombinase binding to the DNA substrates (Figure 27.39). Four molecules of the enzyme
and their associated DNA molecules come together to form a recombination synapse. The reaction begins with the
cleavage of one strand from each duplex. The 5 -hydroxyl group of each cleaved strand remains free, whereas the 3 phosphoryl group becomes linked to a specific tyrosine residue in the recombinase. The free 5 ends invade the other
duplex in the synapse and attack the DNA-tyrosine units to form new phosphodiester-bonds and free the tyrosine
residues. These reactions result in the formation of a Holliday junction. This junction can then isomerize to form a
structure in which the polynucleotide chains in the center of the structure are reoriented. From this junction, the
processes of strand cleavage and phosphodiester-bond formation repeat. The result is a synapse containing the two
recombined duplexes. Dissociation of this complex generates the final recombined products.
27.5.2. Recombinases Are Evolutionarily Related to Topoisomerases
The intermediates that form in recombination reactions, with their tyrosine adducts possessing 3 -phosphoryl
groups, are reminiscent of the intermediates that form in the reactions catalyzed by topoisomerases. This
mechanistic similarity reflects deeper evolutionary relationships. Examination of the three-dimensional structures of
recombinases and type I topoisomerases reveals that these proteins are related by divergent evolution despite little amino
acid sequence similarity (Figure 27.40). From this perspective, the action of a recombinase can be viewed as an
intermolecular topoisomerase reaction. In each case, a tyrosine-DNA adduct is formed. In a topoisomerase reaction, this
adduct is resolved when the 5 -hydroxyl group of the same duplex attacks to reform the same phosphodiester bond that
was initially cleaved. In a recombinase reaction, the attacking 5 -hydroxyl group comes from a DNA chain that was not
initially linked to the phosphoryl group participating in the phosphodiester bond.
III. Synthesizing the Molecules of Life
27. DNA Replication, Recombination, and Repair
27.5. Double-Stranded DNA Molecules with Similar Sequences Sometimes Recombine
Figure 27.37. Recombination. Two DNA molecules can recombine with each other to form new DNA molecules that
have segments from both parental molecules.
III. Synthesizing the Molecules of Life
27. DNA Replication, Recombination, and Repair
27.5. Double-Stranded DNA Molecules with Similar Sequences Sometimes Recombine
Figure 27.38. Holliday Junction. (A) The structure of a Holliday junction bound by Cre recombinase (gray), a
bacteriophage protein. (B) A schematic view of a Holliday junction.
III. Synthesizing the Molecules of Life
27. DNA Replication, Recombination, and Repair
27.5. Double-Stranded DNA Molecules with Similar Sequences Sometimes Recombine
Figure 27.39. Recombination Mechanism. Recombination begins as two DNA molecules come together to form a
recombination synapse. One strand from each duplex is cleaved by the recombinase enzyme; the 3 end of each of the
cleaved strands is linked to a tyrosine (Y) residue on the recombinase enzyme. New phosphodiester bonds are formed
when a 5 end of the other cleaved strand in the complex attacks these tyrosine-DNA adducts. After isomerization, these
steps are repeated to form the recombined products.
III. Synthesizing the Molecules of Life
27. DNA Replication, Recombination, and Repair
27.5. Double-Stranded DNA Molecules with Similar Sequences Sometimes Recombine
Figure 27.40. Recombinases and Topoisomerase I. A superposition of Cre recombinase (blue) and topoisomerase I
(orange) reveals that these two enzymes have a common structural core. The positions of the tyrosine residues that
participate in DNA cleavage reactions are shown as red spheres for both enzymes.
Fly UP