Lecture 15- August 2, 2001

Recombination and Transposons

(Information on retroviruses was not covered in lecture and will not be included on exam 3)

DNA breaks and rejoining can occur

Important for:

  1. Homologous Recombination — meiosis in eukaryotes
  2. Mating type switching of yeast
  3. V(D)L gene rearrangements in T cells — for making antibodies
  4. Insertion of new DNA (extrachromosomal)

Sequences inserted into genome

Happens in transposition and also insertions of retroviral sequences

Homologous Recombination — recombination between similar sequences of DNA

Eukaryotes are diploid — 2 copies of each chromosome

ABC (double helix), after DNA replication becomes ABC/ABC

abc, after DNA replication becomes abc/abc

The two copies of ABC in ABC/ABC are referred to as sisters; the two copies of abc in abc/abc are also called sisters

Homologous recombination can occur between one sister of ABC/ABC and one sister of abc/abc

Example of recombination products (each is a double helix)

ABC

Abc

aBC

abc

 

Homologous recombination allows separation of mutations through independent assortment

Evolution-wise, this is good

Example: Having a and b together may be deleterious, but having A and b may be fine

Breakage and rejoining of chromosomes needs to happen without a nucleotide being lost or gained; don’t want to make a mutation by shifting reading frame

Studies in yeast

Some steps of recombination are conserved in prokaryotes and higher eukaryotes

In yeast, can study recombination at molecular level and isolate the DNA intermediates at different times and see what the DNA looks like

Can also make mutants and see how they’re defective; can determine what proteins mediate what events

 

Recombination in yeast

  1. Double strand break
  2. Resection — chewing back 5’ ends from double strand break
  3. Single stranded 3’ end is left behind, which can invade the other helix and form heteroduplex DNA
  4. Synthesis from 3’ end OH

    5. Synthesize new DNA to fill in gaps

  5. Formation of Holliday Junction

    6. In eukaryotes, there are 2 Holliday Junctions for each break and they can migrate in opposite directions

  6. Resolution — make nicks in DNA

Can result in 1) recombinant chromatids that each contain heteroduplex region OR 2) formation of heteroduplex DNA with NON-recombinant flanking regions

Recombination (drawings not provided See Figure 14.5)

Double strand break — both strands of one helix are cleaved

The recipient is the helix that gets cleaved

Resection — results in free 3’ ends

5’ ends nibbled back, leaving single stranded DNA with 3’ ends

Strand invasion

Formation of heteroduplex region, where one strand from one duplex base pairs with the strand of the other duplex

A 3’ end of the recipient invades the other duplex, displacing one of that duplex’s two strands (i.e. in class example, pink displaces one blue strand)

Note: there may not be perfect base pairing in the heteroduplex region

DNA synthesis

Occurs from 3’ end of recipient that invaded the other duplex; this results in displacement of more of the helix that was drawn in blue. Where new DNA is synthesized will be a perfect match (not heteroduplex)

Ligation reaction

Join newly synthesized DNA to 5’ phosphate of existing recipient (pink) strand

Generate 2 cross-stranded structures, which are the Holliday Junctions

Resolution

Where the cuts are made in the Holliday Junctions determines whether you get recombinant chromosomes or non-recombinant chromosomes

Holliday Junctions See Fig 14.4

Single Holliday Junction

4 node crossed structure — can be seen using electron microscopy

Resolution —

can cut the two parental strands (no cut initially) or can cut the other two strands

In both cases, get 2 double strand structures

Cuts in parental strands

--Create recombinant chromosomes with heteroduplex region in middle

Cuts in other strands

--Does not give recombinant chromosomes although there is a heteroduplex region

Double Holliday Junction

If the two Holliday structures are resolved in the same way (cut the noncrossed strands of both or cut the crossed strands of both) recombinant chromosomes are not generated

If one cut is made on one of the noncrossed strands of one Holliday junction and another cut is made on the crossed strands of the other Holliday junction, then recombinant chromosomes are generated

Thus, resolution of junctions does not always result in recombinant chromosomes

 

Gene Conversion

If not perfect match between strands, it is possible to get DNA mismatch repair that changes heteroduplex DNA

The cell recognizes imperfect match

Fig 14.14 — yeast undergoing meiosis

Usually, get 4 meiotic products

However, some yeast do one round of mitosis after meiosis but before forming spores

In these cases, 8 spores are made, each carrying the genetic information from one of the 8 strands after meiosis

In fig 14.14, when there is no recombination the ratio is 4:4

When recombination occurs and no repair is made, get 3:5 ratio

When recombination occurs and repair does occur, get 2:6 ratio

This effect of divergence from the 4:4 ratio is called gene conversion, in which info on one chromosome is converted into info from another chromosome

Gene conversion can occur in small heteroduplex regions

Transposons — discrete sequences that can move around the genome

IS element in bacteria is an example of a transposon in bacteria

  1. Element usually has inverted terminal repeats
  2. Transposons insert such that there are flanking direct repeats that border the transposon ("direct repeats" refers to the fact that these sequences are oriented in the same direction)
  3. ORF codes for transposase, which cuts DNA and allows rejoining — transposon can move around

Different transposases recognize different inverted terminal repeats

Direct repeats are functions of how the transposase cuts the genome

MOST transposons insert randomly, but some may prefer certain regions (e.g. CG-rich regions)

 

Composite transposons in bacteria contain 2 IS elements with another region in the middle

The middle region between the two IS elements is often involved in drug resistance

The IS elements can hop out, but instead of just the IS elements jumping out individually, the whole unit (the two IS elements with the drug resistance region in between) jumps out, allowing the bacterium to retain its resistance to a certain drug

 

Transposon integration — 2 mechanisms

    1. Replicative transposition

      2. Retain original transposon at original site and get copy of transposon at new site

      Formed through cointegrate

      Need transposase and resolvase (coded by transposon)

    2. Nonreplicative transposition

Breakage and reunion such that transposon moves into new site

Requires only transposase

In both cases, must

1) make cuts in transposon and target and

2) need way to join (and in replicative, resolve) the DNA strands together

Figure 15.10

Nicks are made in transposon

Don’t make nicks and then bring ends of transposon together

Instead, the transposase brings the ends of the transposon together, makes nicks in transposon and then makes nicks in target

Ligate nicked ends of transposon to nicked ends of target

Figure 15.13

Nick in each of the strands of the transposon and target (nicks done by transposase)

Transposase performs ligation event

3’ OH is joined to 5’ phosphate

Ligate transposon to target, which results in crossed structure

Replicative transposition (Fig 15.13)

New DNA synthesis

Can add nucleotide to 3’ end of existing polymer

Now have 2 copies of transposon (hence the name "replicative")

Works for linear pieces too

This forms cointegrate — a single piece of DNA with two direct repeats

Can get recombination between the two repeats (requires resolvase)

When this happens, you generate two circles that each have a copy of the transposon

Nonreplicative transposition (Fig 15.14)

Nicking of donor and target

Ligation of transposon to target to form cross-strand structure

Instead of DNA replication, get 2nd set of nicks in donor (transposon)

Nick in donor generates double strand break and the transposon is now ligated only to the new location

Must repair double strand break

Repair either off the sister or homologous chromosomes to heal the double strand break

Transposon in 2nd piece of DNA (but no longer in original location)