Lecture 2: DNA Replication, Gene Mutations, and Repair

structure of DNA

nitrogenous base

-pyrimidines and purines

sugar molecule

phosphate group

5´ to 3´ direction

What bond connects each nucleotide in the DNA chain?

3’- 5’ phosphodiester bond

3’- 5’ phosphodiester bond function

This linkage results in a free O- at physiological pH, which gives the DNA molecule an overall negative charge

3´OH group on the sugar is required for the addition of the next nucleotide in the growing polynucleotide chain

nucleoside analogues

such as acyclovir and azidothymidine

lack a 3' OH group in their structure

function of 3’ OH in DNA

required for the replicating enzyme to add the next nucleotide in the sequence.

when nucleoside analogues are incorporated into a growing DNA strand, the next nucleotide cannot be added, and chain termination occurs

structure of DNA

Each strand of the double helix is oriented in the opposite direction to the other

B-form, right-handed antiparallel double helix

Hydrophobic bases in the interior

A --T

G --- C

Hydrophilic sugar phosphate exposed to the aqueous

DNA replication

the duplication of the genomic information of a cell

produces 2 semiconservative daughter DNA molecules

both new DNA strands are synthesized in the 5´ → 3´ direction

The leading strand is synthesized continuously

The lagging strand is synthesized in short

-Okazaki fragments

phases of DNA replication

G0 resting phase (senescence)

G1 (growth) phase

S-phase: DNA replication

G2 (growth) phase

M phase: Mitosis- sister chromatids split

semiconservative

one parental and one daughter strand

3 steps of DNA replication

initiation

elongation

termination

initiation definition

occurs at specific nucleotide sequences within the genome known as the origins of replication (Ori)

Ori’s tend to be rich in A-T base pairs

-Prokaryotes have a single origin of replication

-Eukaryotes have multiple origin of replication

Ori

origins of replication

specific nucleotide sequences within the genome where initiation occurs

initiation steps

1. The origin of replication is recognized.

• the origin recognition complex (ORC) binds to Ori

• ORC recruits minichromosome maintenance complex (MCM)

• MCM recruits helicase

2. Helicase unwinds the double helix and forms 2 replication forks

3. Single-stranded DNA binding protein (SSB) prevent reassociation of strands

4. Primase (DNA polymerase α) synthesizes a short RNA primer (5′→3′)

5. DNA polymerase synthesize DNA in the (5′→3′)

• The leading strand is made continuously

• The “lagging strand” is synthesized as small (~1000 nucleotide) Okazaki fragments.

As helicase unwinds, the DNA strands upstream of the fork becomes supercoiled.

Topoisomerase (Gyrase) break the phosphodiester bonds, which relieves the supercoiling.

elongation phase

synthesis of the new DNA strand

DNA polymerases are kept bound to the template with the aid of the sliding clamp proliferating cell nuclear antigen (PCNA)

the RNA primer is no longer needed and must be removed to prevent the formation of a DNA-RNA hybrid

RNAseH (FEN1)

The enzyme responsible for removing the RNA primer in the elongation phase

replication factor C (RCF)

AKA clamp loader

responsible for the initial recruitment of the sliding clamp to the DNA

termination phase

Termination of elongation

occurs when adjacent replication forks meet and resolve each other, or when the replication fork encounters the end of the chromosome.

replication of telomeres

telomerase fills in gap with hexameric repeats

Replicated by telomerase

• RNA-dependent DNA polymerase

• Contains both proteins and RNA template

• RNA template has complimentary sequence to repeating sequence in the telomere

telomeres

ends of linear chromosomes in eukaryotes

contain thousands of tandem repeats of the sequence TTAGGG

fills in gap with hexameric repeats

telomere shortening

As cells age, the activity of telomerase falls progressively.

Loss of telomerase activity leads to the shortening of telomeres, which is considered an indication of cellular aging

Somatic cells has less telomerase activity compared to germ cells and stem cells

Telomere length decreases with every cycle of cell division

telomere sequence is added by telomerase

DNA mutations

Permanent changes in the DNA

Can be inherited if it occurs in germ cells

Potential consequences of DNA mutations:

• Loss of function

• Disruption of the synthesis of a gene (protein)

• No affect on the gene or gene product

cause of DNA mutations

1. internally-derived spontaneous mutations

• depurinations and deamination

2. induced mutations

• X-rays, UV light, carcinogenic chemicals (thymine dimers)

• Defect during DNA replication (trinucleotide expansions)

• Breakage of chromosomes and rearrangements that result from breakage

depurations

Loss of a purine nitrogen base, while the sugar molecule and the phosphate group of the nucleotide remain unchanged.

Missing purine on the template strand may produce a nucleotide deletion in the newly synthesized strand.

deaminations

Cause the conversion of one nitrogenous base to another.

The loss of an amine group from cytosine converts the nitrogenous base to uracil.

When the DNA replication machinery encounters this uracil, it will insert an adenine instead of the cytosine partner guanine, resulting in a G to A nucleotide change at this position in the newly synthesized DNA molecule

point mutations

consequences of depurations and deaminations

single nucleotide change in the DNA sequence

Silent mutation

Missense mutation

Nonsense mutation

insertion/deletion (Frame shift or in-frame mutations)

induced mutation causes

X-ray

ultraviolet light

chemicals

defect during DNA replication

breaking and re-annealing of regions of two different chromosomes

X-ray induced mutations

may cause double strand breaks

• X-rays may generate hydroxyl radicals that react with DNA, altering the structure of the bases or cleaving the DNA strands

ultraviolet light induced mutations

Ultraviolet light may cause pyrimidine dimers

• UV result in the formation of covalent bonds between carbons in adjacent thymines (thymine dimers)

chemicals induced mutations

Chemicals (e.g, Benzo[a]pyrene)

• forms bulky adducts with guanine residues in DNA

defect during DNA replication induced mutation

may result in expansion of the trinucleotides

Breaking and re-annealing of regions of two different chromosomes induced mutations

an cause gross chromosomal rearrangements

repair mechanisms

Nucleotide Excision Repair

Base Excision Repair

Mismatch Repair

Non-Homologous End Joining

Homologous Recombination

nucleotide excision repair

Thymine dimers and bulky adducts are repaired by

Damage (thymine dimers) are detected by XPA, XPB, XPC proteins

Endonucleases (XPC, XPG) cleave the abnormal chain and remove the damaged region

The gap is then filled by a DNA polymerase that adds deoxyribonucleotides, one at a time, to the 3’-end of the cleaved DNA, using the intact complementary DNA strand as a template

The newly synthesized segment is joined to the 5’-end of xthe remainder of the original DNA strand by a DNA ligase

xeroderma pigmentosum

genetic disease in humans associated with mutations in one of the excision repair proteins that is required for the removal of UV-induced thymine dimers

inherited mutation occurs in a member of the XP family of genes, such as XPA, XPB, XPC, etc. Due to impaired excision repair, individuals with this disease are sensitive to light and readily accumulate UV-induced DNA damage, including thymine dimers, upon exposure to sunlight

increased rates of mutation predispose them to developing multiple pigmented growths on the skin and place them at a high risk of skin cancer

base excision repair

used to correct spontaneous mutations introduced during replication caused by deaminations and depurinations

DNA glycosylase cleaves the N-glycosidic bond that joins the damaged base to deoxyribose

The sugar-phosphate backbone of the DNA now lacks a base at this site

An endonuclease cleaves the sugar-phosphate strand at this site

DNA polymerase fills in the gap

DNA ligase joins the newly synthesized segment to the original DNA strand

mismatch repair

During the DNA replication process, DNA polymerase occasionally incorporates the incorrect base, creating a mismatch with the base present on the template strand.

The mismatched bases are removed by the mismatch repair enzyme complex

DNA polymerase then fills in the gap that is produced, incorporating the correct base

Lynch Syndrome AKA Hereditary Non-Polyposis Colorectal Cancer (HNPCC)

hereditary colon cancer caused by mutations in members of the mismatch repair machinery, most commonly MSH2 or MLH1.

When these mutations are present, the cell is unable to repair nucleotide mismatches.

In addition to colon cancer, females with Lynch syndrome are at higher risk for endometrial cancer

Double strand breaks are repaired by…

non-homologous end joining

homologous recombination

non-homologous end joining

double strand breaks are generally produced by ionizing radiation (X-rays, radioactive material)

In somatic cells, doble strand breaks are repaired by non- homologous end joining

• ATM protein is recruited to double strand break.

• Nucleases process the broken ends to form blunt ends

• -In this process some nucleotides in the overhang region may be loss

• Specialized enzymes brings the ends together and rejoined by DNA ligase.

• This repair mechanism is error prone, thus could result in a mutation if it occurs in an expressed gene.

homologous recombination

an error-free method for repairing double strand breaks in DNA

Post-S phase, cells preferentially use homologous recombination.

Since sister chromatids are required for this type of repair, this process must occur after replication, in either G2 or M.

1. ATM is recruited to the double strand breaks by binding to the proteins that recognize the break points and becomes activated.

2. The activated ATM, in turn, activates additional downstream molecules that are necessary for repair of the DNA

Two homologous chromosomes become aligned,

A nuclease generates single-stranded ends at the break by chewing back one of the complementary strands

One of the single strands then invades the homologous DNA duplex by forming base pairs with its complementary strand. A significant number of bases must pair to produce a branch point where one strand from each duplex crosses

The invading strand is elongated by DNA polymerase, using the complementary strand as a template

The branch point migrates as the base pairs holding together the duplexes break, and new ones form

Additional DNA synthesis and ligation completes the repair.

Since sister chromatids are required for this type of repair, this process must occur after replication, in either G2 or M