Cinderella Aquino

3-2-10

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Cell Cycle The cell cycle consists of highly ordered

events that result in the duplication and division of a cell

• M (mitotic) phase - chromosome segregation, cytoplasmic division

• Interphase • G1 (Gap 1) - transition from M to S phase - synthesis of proteins that

would be required during S phase

- G0: Cells that become arrested in G1 (quiescence)

• S (synthetic) phase - DNA synthesis • G2 (Gap 2) - transition from S to M - synthesis of proteins needed

during M phase

Three Key Components of Nucleotides

1. Nitrogenous Base

2. Phosphate

3. Ribose Sugar

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Nitrogenous Bases Purines

Double ring structure Adenine, Guanine

Pyrimidines Single Ring Structure Cytosine & Thymine (DNA

only) Uracil (RNA only)

○ Uracil is not in DNA because cytosine can spontaneously convert into uracil Repair enzymes recognize uracil

in DNA, excise, replaced with cytosine.

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DNA Replication DNA undergoes

semiconservative replication (half of the parent molecule is retained by each daughter molecule)Semiconservative replication

of DNA: The two strands of parental DNA separate, and each serves as a template for synthesis of a new daughter strand by complementary base pairing.

• Check out the silent video at http://www.pbs.org/wgbh/evolution/library/06/3/l_063_02.html

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DNA polymerases catalyze the elongation of DNA chains In the DNA polymerase

reaction, incoming nucleotides are covalently bonded to the 3’ hydroxyl end of the growing DNA chain.

Each successive nucleotide is linked to the growing chain by a phosphoester bond between the phosphate group on its 5’ carbon and the hydroxyl group on the 3’ carbon of the nucleotide added in the previous step (5’-> 3’)

Key Concept:DNA synthesis directional

(5’ to 3’ only)!!!!

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Proofreading is performed by the 3’ -> 5’ exonuclease activity of DNA polymerase

Incorrect nucleotide incorporated about 1 in 100,000 nucleotides. Cell needs process to lower mutation rate

Proofreading mechanismExonuclease activity chews away misincorporated nucleotide

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Eukaryotic and Prokaryotic DNA Polymerases The main types of bacterial DNA Polymerases (I, II, III, IV

& V) DNA Polymerase III is the primary polymerasePolymerase I is responsible for removing RNA primers,

proofreading, DNA repair and filling in gaps left by DNA pol III

The main types of eukaryotic DNA polymerases (, & ) Polymerase found only in mitochondriaPolymerase is involved with initial synthesis of DNA strands off

RNA primersPolymerase synthesizes leading & lagging strands

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DNA replication is usually bidirectional

Origin of Replication Starting site for DNA synthesis

Prokaryotic genomes usually have a single origin of replication Most circular DNA molecule are

replicated in a bidirectional process from a single origin.

This Theta replication occurs in bacteria, mitochondria, chloroplasts and some viruses

Prokaryotic replication origin termed ori Replication Forks

Structure formed when DNA strands are separated at site of DNA replication

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Eukaryotic DNA replication involves multiple replicons

Replicons: units of replication on linear DNA

Typical chromosome may contain several thousand repliconsIn Yeast:

○ Autosomously replicating sequence (ARS element)

Large number of replicons allows eukaryotics to replicate their DNA faster

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Eukaryotic Pre-replication Complex

Initiation process requires formation of pre-replication complex

Origin Recognition Complex (ORC) binds to replication origin

The MCM complex including helicase binds next Requires assistance from helicase loaders

The DNA has been “licensed” for replication The completed complex: Pre-replication

Complex The process of licensing makes sure that

the cells only replicated their DNA only once

First Set of Steps in DNA Replication

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Unwinding DNA double helix requires helicases, topisomerases, and single-strand binding proteins Helicase: unwinds DNA Topoisomerase:

decreases supercoiling caused by unwindingGyrase: a type II

topoisomerase used by bacteria

Single-strand binding proteins: bind to exposed single strands and stabilizes the DNA for DNA replication

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RNA primers initiate DNA replication DNA polymerase MUST have a

3’-OH on the target in order to add a nucleotide DNA polymerase cannot start

synthesis from scratch However, RNA polymerases can

Primase: an enzyme that synthesizes RNA fragments about 10 base pairs long using DNA as a template In bacteria, primase part of a

complex called primosome In eukaryotic cells, primase is tightly

bound to DNA polymerase (initiates DNA replication)

RNA segment is removed when the neighboring growing strand reaches the primer stretch

Prokaryotic vs Eukaryotic Priming

DNA polymerase /

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Leading and Lagging strands

DNA polymerase can only synthesize DNA in the 5’ to 3’ direction

Two types of newly synthesized strands The leading strand is

synthesized continuously in the direction of replication fork movement.

The lagging strand is synthesized in small pieces (Okazaki fragments) backward from the overall direction of replication.

The Okazaki fragments are then joined by the action of DNA ligase.

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Ligase activity: Joining of nickstrands together

A sliding clamp holds moving DNA polymerase onto the DNA

The sliding clamp keeps the polymerase firmly on the DNA when it is moving, but releases it as soon as the polymerase runs into a double-stranded region of DNA Sliding clamp forms a ring-like structure

around DNA The assembly of the clamp around the

DNA requires a clamp loader The clamp loader hydrolyzes ATP as it

loads the clamp on to a primer-template junction.

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Prokaryotes: beta-subunit clampEukaryotes: PCNA

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Summary of DNA Replication-MUST KNOW -

Eukaryotic Replication Fork DNA polymerase in

eukaryotes does not associate into a dimeric complex 2 copies of polymerase remain

separate Replication protein A (RPA)

binds the ssDNA preventing reannealing

Replication factor C (RFC) induces binding of proliferating cell nuclear antigen (PCNA)

Chromatin remodeling proteins are also involved with eukaryotic DNA replication They help move nucleosomes

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Telomerase

Some Key Differences Between Eukaryotic & Prokaryotic DNA Synthesis

1. Eukayotes have multiple origins versus single origin in prokaryotes

2. Eukaryotes undergo DNA synthesis during S phase in cell cycle

3. Eukaryotes remove RNA primers using FEN1 and RNaseH

4. Eukaryotes have telomeres at the ends of their linear DNA molecules, prokaryotic genomes are usually circular and do not have telomeres

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Types of DNA Damage1. DNA replication mismatches

4. Spontaneously loss of nucleosides

Base alteration

Thymine dimer

Double strand break

Mismatches

2. Chemicals (nitrous acid)

3. Radiation

UV

high-energy radiation

Base alteration

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Consequences if deamination (A), or depurination (B), damaged DNA were NOT repaired

Figure 5-50a Molecular Biology of the Cell (© Garland Science 2008)

Deamination of DNA Nucleotides

25Figure 5-50b Molecular Biology of the Cell (© Garland Science 2008)

Table 5-2 Molecular Biology of the Cell (© Garland Science 2008)

Inherited Defects in DNA Repair Cause Known Diseases

Maintaining the integrity of the genome is extremely important for our survival

Mutations in genes involved with DNA repair can increase the risk of cancer and developmental abnormalities

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DNA Repair Mechanisms Translesional synthesis:

DNA synthesis of new DNA across regions in which the DNA template is damagedDNA polymerase eta can

catalyze DNA synthesis across regions with thymine dimers

Excision repair pathways Base excision repair: repair

single damaged basesNucleotide excision repair:

repair major distortions in DNA double helix○ Pathway that corrects thymine

dimersMutations in pathway causes

Xeroderma pigmentosum

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Uracil-Glycosylase Removes Uracil From DNA When DNA is damaged through deamination

reactions, cytosine is converted to uracil Error repaired by uracil-DNA glycosylase Damage repair helps explain why DNA contains

thymine instead of uracil If uracils were normally found in DNA then DNA repair would

not be able to distinguish normal uracils from deamination generated uracils

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AP Endonucleases

• Two Key Roles of AP Endonuclease: • Repair nucleotides missing bases caused by

spontaneous events, such as depurination • Repair nucleotides missing bases caused by

target repair process, such as by uracil glycosylase

Nucleotide Excision Repair (NER)NER corrects problems such as pyrimidine dimers

Steps in NER1. Identification of the mismatched or mutated DNA strand

2. Nick the mismatched DNA or mutated strand by endonuclease

3. 5’-3’ DNA polymerase fill the gap,

4. DNA ligase forms phosphodiester linkage

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NER Diseases Human genetic disorders associated with defects on nucleotide excision repair:

Xeroderma Pigmentosum (XP), Cockayne syndrome, Trichothiodystrophy XP characterized by sun sensitivity, ocular involvement, > 1000-x

increased risk of cutaneous and ocular neoplasms. ~50% of affected individuals demonstrate acute sun sensitivity from early infancy, acquiring severe sunburn with blistering or persistent erythema on minimal sun exposure; marked freckling of sun-exposed areas in a child before age two years is typical of XP

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Mismatch Repair Normal, undamaged, but

mismatched bases bind proteins of the mismatch repair system.

In bacteria, these proteins recognize the older, parental strand because it is methylated and replace a segment of newly synthesized (and unmethylated) DNA containing the mismatched base.

The mechanism for distinguishing between parental and newly synthesized strands in humans is not as well understood. Scan for nicks in the new strand

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Prokaryotes

Eukaryotes

Human Mismatch Repair and HNPCC

HNPCC: Hereditary Non-Polyposis Colon Cancer

~40%~50%

~10%

Adapted From Peltomaki (2001) Human Molecular Genetics 10:735 33

• Hereditary nonpolyposis colon cancer (HNPCC) results from mutations in genes coding for proteins involved in mismatch repair

• Most common mutations: • MLH1 and MSH2

Mutation ‘hot spots’ The occurrence of point mutations in the genome is not

necessarily a random event. 30% of point mutations in known inherited diseases, involve C → T

transitions (G → A, in the opposite strand), at sites containing CG dinucleotides (the so called CpG islands)

The CG doublet represents a true ‘hotspot’ for mutation in human genome.

Transitions occur more frequently at CpG islands, because the cytosine is prone to methylation at position 5, & spontaneous deamination of 5-methylcytosine to thymine follows [ C→ 5-MethylC→T ] ○ C→T transition in one strand is matched in the opposite strand by G →A transition

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Repair of Double-Strand DNA Breaks Double-strand DNA Breaks are repaired by nonhomologous end-

joining or homologous recombination Nonhomologous end-joining uses a set of proteins that bind to the

ends of the two broken DNA fragments and joins them together Error-prone

○ A typical somatic cell in a 70-year old had over 2,000 end-joining repairs

Homologous recombination The intact chromosome acts as a template to guide repair of the damaged

chromosome Breast Cancer genes BRCA1 & 2 involved with this pathway

Homologous RecombinationHomologous Recombination Repairs Spontaneous Breaks in DNA and Those Induced in Meiotic Crossing-over

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Nonhomologous End-Joining

Ku heterodimers grasps the broken chromosome ends and help recruit other proteins to fix the break

Hereditary Breast and Ovarian CancerBreast Cancer Susceptibility Genes BRCAs BRCA1 and BRCA2 are tumor suppressor proteins

Abnormal BRCA1: dominant susceptibility gene- confers high risk of breast and ovarian cancer (Type 1).○ BRCA1 breast cancer affects 1:800 women in the U.S. ○ Jewish women of eastern European descent, the risk is

1:100 BRCA2 gene confers a high risk of breast cancer, not an

elevated risk of ovarian cancer. (Type 2).Mutations also increase risk of prostate, pancreatic, gall

bladder, and male breast cancers.

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