Biomedical Sciences: DNA Organization, Replication and Repair (Lecture 1; Smoczer)

What are nucleotides?

Monomeric units of DNA

• Nitrogenous base

• Sugar

• Phosphate

Purines: Nitrogenous Bases

• Adenine (A)

• Guanine (G)

Pyrimidines: Nitrogenous Bases

• Cytosine

• Thymine

What is each DNA molecule formed by?

• Two polynucleotide bonds

• Hydrogen bonds between bases

How many hydrogen bonds are between A and T?

2.

How many hydrogen bonds are between G and C?

Three.

What is DNA denaturation?

Heat/alkali destruction of hydrogen bonds; the 2 strands separate since phosphodiester bonds are resistant.

Renaturation (reannealing):

Dissociation is reversible by reversal of denaturing conditions.

What is Melting Temperature? (Tm)

Temperature at which 50% of DNA is single stranded.

(Depends on the base composition).

What is Hybridization?

The tendency of single-stranded DNA to bind complementary sequences of DNA or RNA.

Types of DNA:

• Mitochondrial

• Nucleic

Genetic Information properties:

• Inherited (Replication)

• Expressed (Transcription & Translation)

Characteristics of Mitochondrial DNA

• Circular, double-stranded (endosymbiotic theory)

• Approx 1% of total cellular DNA

• Lacks histones and is GC rich

• 37 genes

• Derived exclusively from the mother.

• Marker for human ancestry (no recombination)

What do mitochondrial DNA look like?

They are circular & double-stranded.

How many genes do mitochondrial DNA have?

• 13 (energy production)

• 24 (for rRNA and tRNA)

How many mitochondrial DNA genes are for energy production?

13.

How many mitochondrial DNA genes are for rRNA and tRNA?

24.

Mitochondrial DNA are derived exclusively from where?

The mother.

Nucleic DNA is mostly ___.

Non-coding (98%).

How long is Nucleic DNA?

Extremely long (approx 6ft/cell) encoding ~22000 genes.

T/F: Nucleic DNA is mostly coding.

False. It is mostly non-coding.

What are centromeres/

Constricted region of the chromosomes.

• Attachment site for the spindle microtubules

• Required for correct mitosis

What is the attachment site for the spindle mirotubules?

Centromere.

What are Telomeres?

Caps at the ends of chromosomes.

• Repeats of TTAGGG (1000x)

• Shortened with each cell division.

What happens with telomeres after each cell division?

They become shorter.

What is Chromatin?

Chromosomes are formed by super-compacted DNA-protein complexes.

Characteristics of Euchromatin

• LESS condensed

• On chromosome arms

• Unique sequences

• Many genes

• Transcription occurs often

Characteristics of Heterochromatin:

• More condensed

• At centromeres, telomeres, and other specific places

• Repeated sequences

• Few genes

• Infrequent transcription

What are nucleosomes?

Structural chromatin unit; histone octamer core + dsDNA wrapped 2x around the core

What is the histone octamer core compoed of?

Two molecules of each; H2A, H2B, H3, H4

What does “beads-on-a-string” refer to?

Linker DNA + histone H1 → connect to adjacent nucleosomes.

What are chromatin fibers?

Supercoiled nucleosomes. (solenoid, tubular coils)

What are chromosomes?

Compaction of chromatin fibers.

DNA Synthesis is required for:

Replication and repair.

Cell Cycle:

Sequence of ordered events, during which proliferating cells replicate their chromosomes and separate into daughter cells.

What is the S phase?

DNA replication.

What is the G2 phase?

DNA repair.

How long does genome duplication take in a cell?

Approximately 9 hours.

Origins of Replication:

DNA replication begins at specific sequences

Origins of Replication: Euakaryotes

100 / chromosome

Origins of Replication: Prokaryotes

ONLY 1 in prokaryotes.

DNA replication is ____ and ____.

Semiconservative and bidirectional.

What does semiconservative DNA replication mean?

Each new dsDNA contains one parental and one daughter strand.

What does bidirectional mean?

In both directions from the origin.

Replication Fork

The Y-shaped region where synthesis occurs.

What does Helicase do?

Breaks hydrogen bonds and separates the two strands.

What does DNA topoisomerase do?

Relaxes coils and unwinds DNA ahead of replication fork.

What are Single-stranded binding proteins? (SSBP)

Attaches to each strand to prevent reannealing.

What is Primase?

A subunit of DNA polymerase.

(Synthesizes an RNA primer, approx. 15nt by copying the parental strand)

What does DNA polymerase do?

Adds dNTPs to the free 3’-OH of the RNA primers.

(Always leaves a free 3’-OH to accept more dNTPS.)

How does DNA polymerase read parental DNA?

from 3’ → 5’

How does DNA polymerase synthesize daughter DNA?

5’ → 3’

How does DNA polymerase remove DNA primer?

Has 5’ → 3’ exonuclease activity to remove the RNA primer.

Proofreading Activity of DNA Polymerase:

Uses 3’ → 5’ exonuclease activity excises mismatched nucleotides, which allows for normal continuation of synthesis.

What does DNA Ligase do?

Joins the free 3’-OH group and the free 5’-phosphate group to form a phosphodiester bond.

What does the Leading strand do?

• Runs 3’ → 5’ towards the replication fork.

• Is replicated continuously from an RNA primer by DNA polymerase.

What does the Lagging strand do?

Runs 3’ → 5’ away from the replication fork.

How is the lagging strand replicated?

Replicated discontinuously from multiple RNA primers by DNA polymerase to form Okazaki fragments (200nt).

How are primers removed in the lagging strand?

Primers are removed by 5’ → 3’ exonuclease and gaps are filled.

The ends of adjacent fragments in the lagging strand are joined by:

DNA Ligase.

What are Okazaki fragments?

Short, newly synthesized pieces of DNA that form on the lagging template strand during DNA replication.

What is the ‘end-replication problem’?

Removal of RNA primer from the 3’-end of the daughter strand during lagging strand synthesis leads to shorter, newly synthesized DNA strands.

Additional rounds of replication = progressive shortening of telomeres.

What is Telomerase?

Preserves the length of telomeres by copying a small number of repeats, after which the complex moves forward to the 3’-end of the overhand and repeats the process.

What kind of enzyme is Telomerase?

A ribonucleoprotein. (combines both protein and RNA parts)

Telomerase (TERT gene):

Reverse transcriptase

Telomerase (TERC gene):

Provides the RNA template that the telomerase enzyme uses to protect and maintain the ends of chromosomes.

TERC gene: RNA template

3-20kb hexonucleotide repeat (3’-AAUCCC-5’)

What kind of cells do NOT express telomerase?

Somatic cells.

(causes cell aging and progressive shortening of chromosomes)

In what cells is telomerase expressed?

• Germline cells

• Cancer cells

What causes DNA damage?

• Replication errors

• Spontaneous

  • Alkylation

  • Depurination

  • Deamination

• Mutagen-induced damage

Fidelity of replication:

Errors approx. 1 in every 10^9 - 10^10 nt

(very low but still possible)

What is endogenous damage?

Mismatched bases during replication.

What is exogenous damage?

DNA damaged by mutagens.

T/F: Hundreds of thousands of mutagenic lesions are repaired daily.

True.

What are mutations?

Permanent changes that are undetected/unrepaired.

T/F: Errors are mutations.

FALSE; Errors are not mutations.

Endogenous mutations:

Free radicals;

(Metabolic byproducts, spontaneous chemical instability, replication machinery failures)

Exogenous mutations:

• Alkylating agents

• Radiation (e.g. ionizing radiation, X-rays, UV)

• Chemicals (e.g. pollutants, pesticides, plant and microbial toxins, smoking, chemotherapeutics)

What are Carcinogens?

Mutagens that induce normal cells to become cancerous.

Examples of Singe-strand damage:

• Direct damage reversal

• Excision repair

  • Base excision repair (BER)

  • Mismatch repair)

  • Nucleotide excision repair (NER)

Examples of Double-strand damage?

• Recombination repair

  • Homologous recombination (HR)

  • Non-homologous end joining (NHEJ)

Direct Damage Reversal:

• Simplest mechanism = repair done without cuts in the DNA backbone

• Mostly for removal of drug-induced (chemotherapy) alkylation of guanine (06-methylguanine)

What are methyl groups removed by?

Methyltransferase.

Why is the methylation of guanine a wasteful process?

Methylated enzymes becomes inactive → one enzyme is needed for each methyl group removed.

Generic DNA excision repair pathyway:

• Common steps:

  • Direction of damage

  • Excision of damaged DNA by endo-/exonucleases

  • Replacement of excised region by DNA polymerase

  • Nick sealing by DNA ligase

What is Base Excision Repair (BER)?:

Repairs small distortions in DNA caused by damaged bases.

• Spontaneous

• After exposure to alkylating or oxidizing agents

• Sugar-phosphate backbone remains intact

What makes BER spontaneous?

• Depurination: (>1000/ day)

• Deamination: (C → U) (>100/day)

  • Deamination of methylated C → T escapes repair

What is Mismatch Repair? (MMR)

Removes and repairs mismatched, but undamaged bases added during replication (escape DNA polymerase proofreading).

(Corrects small insertions or deletions)

What strand is used as MMR template?

Parental strand.

What do defects in MMR lead to?

• Lynch syndrome (AD)

• Colon cancer (15% all colon cancers, and hereditary non-polyposis colon cancer)

How is MMR triggered?

Mismatched bases in newly synthesized DNA produce distortions.

Newly synthesized strand recognized by presence of nicks.

What is Nucleotide excision repair? (NER)

Detects and corrects bulky damage that distort the DNA double helix → replication block.

What inhibits NER?

• Cigarette smoke - benzo(a)pyrene-guanine adducts.

• UV radiation - adjacent pyrimidine dimers (T-T)

What does unresolved T-T dimers result in?

UV-induced skin cancers.

Defects in NER can cause:

Xeroderma pigmentosum (AR).

Repair of double-strand breaks:

Ionizing radiation, ROS, and inadvertent action of nuclear enzymes induce double-strand breaks (DSBs) in the DNA.

What is the MAIN cause for genomic instability and large mutations?

Double-strand breaks (DSBs).

What is homologous recombination?

Information from the sister chromatid is used as template to replace the missing region.

(More complex, but more precise)

When does homologous recombination occur?

Mostly during the S-phase of the cell cycle.