Chapter 11 - DNA Replication and Recombination

Bacterial Defense Mechanisms

• Restriction enzymes and DNA methylation (self): ‘innate’

• CRISPR/Cas: ‘adaptive’

Restriction enzymes

• Cut DNA at specific recognition sequences

  • A restriction enzyme is a endonuclease whose activity is restricted to a specific DNA recognition sequence.

    • The DNA sequences recognized are usually palindromic

    • The cleavage can be straight across the double-helix, leading to blunt end, or staggered across the helix leading to sticky ends

  • DNA fragments are produced by DNA restriction

Palindromic DNA

Ex. 5’…GAATTC…3’

3’…CTTAAG…5’

Restriction enzymes

• Most recognition sequences are palindromic, and restriction enzymes often cleave these sequences in an offset manner

Restriction and Electrophoresis

• After separation, DNA or RNA fragments are visualized by staining with the dye ethidium bromide and examination under ultraviolet light

DNA Sequence analysis

• This is the dideoxy chain termination sequencing, developed by Fred Sanger (receiving his 2nd Nobel prize).

  • This method is based on in vitro DNA synthesis. Tubes have all four dNTPs and a small amount of one of the ddNTP (e.g. ddATP or ddGTP, etc.).

  • The dideoxy nucleotides lack the hydroxyl group on both the 2’ and 3’ carbons. No more extension of DNA molecule!

  • In the first variation of this method, a radioactive nucleotide is added to the sequencing reaction for detection. In the second iteration, fluorescent nucleotides were used for detection.

DNA Replication Learning Goals

• Understand how we came to know that DNA replication is semi-conservative

• Understand the molecular basis of DNA replication

  • Know the enzymes and their roles

  • Appreciate how DNA lends itself to copying, but also recognize some of its quirks and shortcomings

A model of DNA replication

The Meselson-Stahl experiment

• The Meselson-Stahl experiment demonstrated that:

  • DNA replication is semiconservative…

  • … or, in other words, each new DNA molecule consists of one old strand and one newly synthesized strand

Comparing the original figure to the one in the textbook

Semi-conservative replication is also true in eukaryotes

Taylor-Woods-Hughes experiment looked at radioactive label in root tip cells

image is a, there is b on recording plz update

Tasks during DNA replication

• Many Complex Tasks Must Be Performed during DNA Replication

  • The replication origin(s) must be recognized

    • in bacteria there is one origin; in eukaryotes, there are many

  • There are seven key issues that must be resolved during DNA replication:

    • unwinding of the helix

    • reducing increased coiling generated during unwinding

    • synthesis of a primer for initiation (5’ to 3’ direction)

    • DNA synthesis and proofreading (to prevent errors)

    • discontinuous synthesis of the second strand

    • removal of the RNA primers

    • joining of the gap-filling DNA to the adjacent strand

Genetic approaches were used to find the genes necessary for replication

A number of genes involved in DNA replication have been identified by conditional mutations

• These mutants survive (replicating DNA and completing cell division) at low permissive temperatures but show phenotypes at higher temperatures

Don’t memorize gene names, just think about the proteins that come from them

The origin of replication

• Prokaryotes have one origin of replication (oriC)

  • E. coli has 4.6 × 10^6 bp in one circular DNA molecule.

  • Two replication forks move away from the origin, and terminate on the opposite side of the genome.

• The origin is recognized by the DnaA protein.

Bidirectional is from both forks going around

Recognition of the origin

• In E. coli, the replication origin, oriC, is bound by 10-20 copies of DnaA protein in the “9-bp” repeat region

• This wrapping denatures the adjacent AT-rich DNA, stabilized by SSB (single-stranded binding protein), which allows the DnaBC helicase to bind and further unwind the DNA helix.

Recognition of oriC

The unwinding of the AT-rich region is the crucial step in the initiation of most origins, prokaryotic and eukaryotic ones… Initial binding [of DnaA-ATP to a high affinity site] …then serves as an anchor for the cooperative binding of ATP-DnaA to [low affinity sites in oriC] … DnaA bends the DNA by 40° upon binding [and the] topological stress in this complex unwinds the DNA in the AT-rich region… Cooperative binding to the double-stranded AT-rich region is therefore presumably the limiting step in the initiation reaction, followed by unwinding [and helicase interaction for further unwinding]

Replication: DNA synthesis

Priming the reaction to provide a 3’-OH for synthesis to start. The catalytic reaction is a nucleophilic attack on the incoming phosphate to form a phosphoester bond. Unwinding of a helix creates “knots” in the structure.

DNA synthesis

• Requirements for DNA synthesis

  • DNA template (in pink)

  • Primer to provide a 3’-OH (in blue)

  • DNA-dependent DNA polymerase

  • Deoxynucleotides(dNTPs)

Formation of a phosphodiester linkage in a DNA synthesis reaction

• The incoming base (purple) is a nucleotide triphosphate.

• The 3’-OH is a nucleophile, attacking the phosphorus atom of the α-phosphate group.

• Pyrophosphate is the leaving group. Pyrophosphate is hydrolyzed to two inorganic phosphate groups, making the bond formation non- reversible.

Tension and topology

When you uncoil a helix, it causes overcoiling ahead of the fork

DNA Topology - Twisting and Writhing

• Advanced replication forks and strand separation →

• DNA overwinding →

• Supercoiling

• i.e Tw + Wr would stay constant during replication unless an enzyme counteracts this physical property

More tension and topology

DNA topoisomerases, like gyrase, create nicks in the phosphodiester backbone. This lets the molecule unwind to release the tension

Mechanism of gyrase (a Type 1 Topoisomerase)

DNA replication causes torsional stress of DNA that is counteracted by DNA gyrase

DNA configuration in the absence of DNA gyrase; the overwound DNA inhibits further replication

Primers are required to start synthesis

• DNA polymerase can add nucleotides to a 3’ end

  • It can extend a growing DNA chain, but it can’t initiate one

  • So, what does it do in the absence of a 3’ end? Nothing! (really!)

• Primase, an RNA polymerase, will synthesis an RNA primer on a DNA template

  • Primase binds to the DnaB helicase and SSB

  • It synthesizes a short run of RNA; this will serve as the primer

  • DNA polymerase can take over from there

What’s happening at the replication forks?

• The DNA helix is being unwound (helicase) and stabilized (single-stand binding protein)

• Torsional stress is being relieved (by gyrase) as the fork moves forward

• DNA polymerase is catalyzing the polymerization of the newly forming DNA strand

  • Nucleotides are added at the 3’ end of the growing molecule

Leading and lagging strands

• Primase synthesizes a short RNA primer on the ssDNA

• DNA polymerase extends from these primers with DNA, in the 5’ to 3’ direction. The direction of synthesis is important.

• As more DNA is unwound (by helicase), the leading strand has continuous synthesis towards the moving fork, but new primers have to be made on the opposite strand: discontinuous synthesis

Replication from primers at the forks

• Initiation bubble forms (DnaA, helicase, SSB)

• Primase forms RNA primers

• DNA polymerase is loaded on DNA

  • (by the “Gamma complex”)

  • Primase leaves

• DNA polymerase is bound to DNA

  • (The beta-clamp subunit is responsible)

  • increases “processivity”; the extent of DNA synthesis is longer

• Synthesis begins on leading strand

DNA polymerases

• DNA synthesize (polymerize) DNA in the 5’ to 3’ direction

  • Prokaryotic DNA polymerase III is responsible for most of the DNA replication in prokaryotes

    • DNA polymerase I fills in gaps (see below)

    • DNA pol II is important for DNA repair

• But…., they can do more than that

  • Can also reverse course and remove nucleotides (exonuclease activity) in the 3’ to 5’ direction

    • “Proofreading” capacity decreases the error rate by a factor of 100

  • Prokaryotic DNA Polymerase I can remove nucleotides in the 5’ to 3’ direction

    • Responsible for removing RNA primers and replacing them with DNA by extending the adjacent DNA strand

Leading and lagging strands again

DNA polymerase I removes the RNA and replaces it with DNA

DNA ligase joins all of the fragments together on the lagging strand

DNA polymerase I removes the RNA primer

• DNA synthesis leaves a nick between DNA and RNA

• Pol I binds to nick 5’→3’ Exonuclease activity removes RNA primer

• 5’→3’ DNA polymerase activity synthesizes DNA to fill gap

• DNA Ligase comes in and seals the nick (creates a phosphodiester bond)

E. coli DNA polymerases

E. coli DNA polymerases pt2

Many complex tasks must be performed during DNA replication

• Seven key replication issues are solved by key enzymes

  • unwinding of the helix (DnaA initially, helicase, SSB)

  • reducing increased coiling generated during unwinding (topoisomerases, gyrase)

  • synthesis of a primer for initiation (primase, aka an RNA polymerase)

  • DNA synthesis and proofreading (to prevent errors) (DNA polymerase III, DNA polymerase I)

  • discontinuous synthesis of the second strand (DNA pol III, DNA pol I)

  • removal of the RNA primers (DNA pol I)

  • joining the DNA fragments (DNA ligase)

Eukaryotic DNA synthesis is similar but more complex than DNA synthesis in prokaryotes

• Large size of genome requires coordination of the replication initiation at many different replication origins.

  • 16 chromosomes and ~350 replication origins in yeast

  • 46 chromosomes and ~80,000 replication origins in mammals

• Linear chromosomes present a replication problem at the ends.

  • Telomere addition by telomerase extends chromosomes

Replication origins - eukaryotic

• In eukaryotes, there are multiple replication origins.

  • There are about 80,000 origins in a mammalian genome; 250-400 in the yeast genome. This allows all of the genome (in humans, 6 billion base pairs in total) to be replicated in a few hours.

  • A replicon is the length of DNA that is replicated following one initiation event at a single origin.

  • Replication proceeds bidirectionally from each origin.

  • Like bacteria, eukaryotes have origin recognition proteins, helicases, and single strand binding proteins that play the same molecular roles. You do NOT need to know their names.

Eukaryotic DNA polymerases

• In eukaryotes, there are several more polymerases.

  • Pol a and b are the major forms of the enzyme involved in initiation and elongation.

    • Pol a possesses low processivity, a term that reflects the length of DNA that is synthesized by an enzyme before it dissociates from the template. Pol a functions in synthesis of the primers during initiation on the leading and lagging strands.

    • Polymerase switching occurs, and Pol a is replaced by Pol b for elongation.

The End (The Telomere)

• Replication problems at the ends of linear chromosomes

• Structure stabilizes ends, protecting them from exonucleases

End replication problem

• Lagging strand synthesis at the end of the chromosome is a problem because once the RNA primer is removed, there is no free 3'-hydroxyl group from which to elongate.

  • This is a big problem because there is a loss of genetic material at every cell division and with every generation (see gap b in figure).

  • Telomeres are the eukaryotic solution to the replication problems at the ends of chromosomes.

Telomere

Telomeres are specific sequences located at the ends of linear chromosomes

• They consist of long stretches of short repeating sequences.

• They provide a means to copy fully the lagging strand.

• They preserve the integrity and stability of chromosomes (preventing recombination).

Telomerase

• Telomerase directs synthesis of the telomere repeat sequence to fill the gap.

  • This enzyme is a ribonucleoprotein with an RNA molecule that serves as the template for the synthesis of its DNA complement.

  • In other words, telomerase is an RNA-dependent DNA polymerase (a reverse transcriptase!)

Telomerase and synthesis of the repeats a)

Telomerase and synthesis of the repeats b)

Telomerase and synthesis of the repeats c)

Telomerase and synthesis of the repeats d)

More repeats are added than are lost when primer is removed

Telomerase adds bases in groups of 6

Telomeres adopt unusual structures: T-loops and G-quadruplexes

T-loops and G-quadruplexes are thought to prevent DNA repair pathways from detecting ends as double strand breaks and degrading or fusing the chromosome ends

Telomeres adopt unusual structures: T-loops and G-quadruplexes

Telomeres vary in length

• Telomere length varies greatly between species (the double stranded repeat region)

  • ~ 300 base pairs in yeast → 2-50 kilobases in humans

• Telomere length varies with age

• Human telomeres shorten by ~70 bp/year (depending on tissue)

• Why? Telomerase is shut off in somatic cells!

• Provides a limit to number of divisions (Hayflick limit – 50-70 cell divisions)

Telomere loss protects against cancer

Shorter telomeres are associated with poorer health and aging

• Telomeropathies are diseases caused by mutations affecting telomere length

  • (too short → faster age-related disease)

• Why aren’t longer telomeres selected for to ameliorate these effects?

E. coli DNA polymerases

Extra sides

E. coli DNA polymerase III is the main enzyme for DNA replication

Extra slides

E. coli DNA polymerases

Extra slides

Rate of telomere shortening is predictive of species life span

Extra slides

E. coli - replication at the fork

Learning goals for recombination

• Understand the molecular basis of recombination

  • Know the single-strand-break repair model; importance of the Holliday junction (from the book)

  • Understand how DNA does it: the double strand break model (see in Ch. 15 Mutation & Repair)

  • Important: Recombination is precise – between base pairs; at chiasmata in meiosis.

• Gene conversion – it is a consequence of the repair of small differences from heteroduplex formation

Recombination and it’s importance

• During meiosis, recombination puts together good alleles at different loci (evolutionary reasoning)

  • Might be the benefit of sexual reproduction

  • Used to map locations of genes relative to each other

• In bacterial, necessary for Horizonal Gene Transfer

• In meiotic cells and mitotic cells, it can repair double strand breaks, fixing or causing inversions, translocations

  • Template mediated (from the sister chromatid)

  • An alternative is non-homologous end-joining (NHEJ)

• In immunology, recombination is responsible for the diversity of immunoglobulins found in B cells

  • This is somatic recombination: same mechanism (i.e., same dance of the DNA strands) but in somatic cells

Recombination in meiosis

Homologous recombination in yeast

what is a holliday junction

Phage lambda integration (prophage)

• Integration:

  • Lysogenic phage (like λ) do this to make a prophage; shown is integration. Excision is the reverse process with the same enyzmes.

  • Integration of the F plasmid to make an Hfr.

  • Phage P1 integrates using loxP sites (instead of attP or attB) to make a lysogenic prophage; the name of the catalytic enzyme is CRE.

Cre-Lox used in mouse genetics

another slide in presentation to look at

Shows Cre LoxP mouse

Holliday juncture and recombination model

Holliday structure and recombination

Holliday steps in detail

• Notice the linkage:

  • A-B and a-b

• Notice the polarity of the strands

  • An enzyme nicks the DNA; i.e., cuts one phosphodiester bond on one of the strands.

Holliday in detail

• Proteins mediate strand exchange

  • Helicase activity is required

Holliday more in detail

• Ligation connects the strands

Branch Migration

• Branch migration moves the region of heteroduplex formation.

  • Important to make sure molecules are homologous.

  • Note that if there are small, single nucleotide differences, there would be mismatches. These would be repaired and could lead to ‘gene conversion’.

Branch migration through a Holliday junction

Reorientation of the structure

More image

Resolution of the Holliday Junction

• The crossed DNA strands must be separated from each other - i.e. the structure must be resolved

• Cut and then re-ligate

Formation of the recombinant DNA

• Note the association of A with b, and a with B

• A Recombinant!

• What would happen if cut was horizontal instead of vertical?

Potential gene conversion

What would happen if cut was horizontal instead of vertical?

Note the association of A with B, and a with b with heteroduplex in middle

Holliday structure and recombination