DNA Replication and Repair

Key Concepts of DNA Duplication

• DNA serves as the genetic material.
• DNA replication and repair involve many proteins working together.
• A chromosome contains DNA packed with proteins.

DNA as Genetic Material

• Hereditary information is encoded in DNA and directs development of traits.
• Identifying molecules of inheritance was a major challenge in the early 20th century.
• Genes are located on chromosomes; DNA and protein were potential candidates for genetic material.
• The role of DNA in heredity was discovered through studies on bacteria and viruses.

Griffith's Experiment

• Frederick Griffith's 1928 research marked the discovery of DNA's genetic role.
• Griffith experimented with pathogenic (S strain) and harmless (R strain) bacteria.
• Transformation is a change in genotype/phenotype due to assimilation of foreign DNA.
• The macromolecule responsible was not immediately determined to be DNA or protein.

Bacteriophages

• Further evidence for DNA as genetic material came from bacterial virus studies.
• Bacteriophages/phages are viruses commonly used in molecular genetics research.
• Viruses have DNA (or RNA) within a protective protein coat.

Hershey-Chase Experiment

• Alfred Hershey and Martha Chase demonstrated in 1952 that DNA is the genetic material of the T2 phage.
• Their experiment showed that, of the T2 components, only one enters and infects E. coli cells.
• Conclusion: injected phage DNA is responsible for genetic information.

Chargaff's Rules

• DNA consists of nucleotide polymers with nitrogenous bases, sugar, and phosphate groups.
• In 1950, Erwin Chargaff reported that DNA composition varies between species.
• Chargaff's rules: DNA base composition varies between species and the number of A and T bases are equal, as are G and C bases.
• The basis for Chargaff's rules was not understood until discovery of the double helix structure.

Discovery of DNA Structure

• After DNA was accepted as genetic material, the challenge became determining its structure.
• Maurice Wilkins and Rosalind Franklin used X-ray crystallography to study DNA structure.
• Franklin's X-ray images enabled Watson to deduce DNA's helical nature and strand number.

DNA Model

• In 1953, James Watson and Francis Crick proposed the double-helical model for DNA structure.
• Watson and Crick's double helix model conformed to X-ray data and DNA chemistry.
• Franklin concluded that sugar-phosphate backbones are on the exterior, with paired bases inside.
• Watson's model showed backbones are antiparallel, running in opposite directions.

Base Pairing

• Watson and Crick initially thought bases paired "like with like," but this didn't yield a consistent width.
• Purine-pyrimidine pairing resulted in a uniform width matching X-ray data.
• Adenine (A) pairs only with thymine (T), and guanine (G) pairs only with cytosine (C).
• The Watson-Crick model explains Chargaff's Rules where the amount of A = T and C = G.

Mechanism of DNA Replication

• Watson and Crick's base pairing suggested a copying mechanism for genetic material.
• DNA strands are complementary, so each acts as a template for building a new strand during replication.
• In replication, the parent molecule unwinds, and new daughter strands are built using base-pairing rules.
• The semiconservative model produces daughter molecules, each with one original and one new strand.

Competing Models of Replication

• Competing models were the conservative model (parent strands rejoin) and the dispersive model (each strand is a mix of old and new).

Meselson-Stahl Experiment

• Matthew Meselson and Franklin Stahl's experiments supported the semiconservative replication model.
• Nucleotides of old strands were labeled with heavy nitrogen, while new strands were labeled with light nitrogen.
• First replication resulted in a hybrid DNA band, disproving the conservative model.
• Second replication yielded both light and hybrid DNA, eliminating the dispersive model.

Origin of Replication in Prokaryotes

• Copying DNA is very accurate and rapid, involving many enzymes and other proteins.
• Replication starts at origins of replication, where two DNA strands separate, forming a bubble.

Origin of Replication in Eukaryotes

• Eukaryotic chromosomes have hundreds or thousands of origins of replication.
• Replication proceeds in both directions from each origin, until the entire molecule is copied.

Replication Fork

• Replication fork: a Y-shaped region where new DNA strands elongate at the end of each bubble.
• Helicases are enzymes that untwist the double helix at replication forks.
• Single-strand binding proteins stabilize single-stranded DNA.
• Topoisomerase corrects overwinding ahead of replication forks by breaking, swiveling, and rejoining DNA strands.

Primase

• DNA polymerases catalyze elongation of new DNA at the replication fork.
• DNA polymerases can't start synthesis of a polynucleotide; they can add nucleotides only to an existing 3' end.
• Primase synthesizes a short RNA primer, adding RNA nucleotides one at a time.
• This short primer allows DNA replication to begin.

DNA Polymerase

• Each nucleotide added to the growing strand is a deoxynucleoside triphosphate (dNTP), which is similar to ATP.
• dNTPs have deoxyribose, while ATP has ribose.
• As nucleotides join the growing strand, pyrophosphate is released.
• Two main types of DNA polymerases: DNA polymerase III (main builder) and DNA polymerase I (replaces the RNA primer with DNA).
• Elongation rate: 500 nucleotides per second in bacteria, 50 per second in human cells.

Direction of Polymerization

• The antiparallel DNA structure affects replication.
• DNA polymerases add nucleotides only to the free 3' end of a growing strand, elongating in the 5' to 3' direction.
• A leading strand is synthesized continuously, moving toward the replication fork.
• To elongate the lagging strand, DNA polymerase must work away from the replication fork.
• The lagging strand is synthesized in segments called Okazaki fragments, which are joined by DNA ligase.

Leading Strand Synthesis

• After an RNA primer is added, DNA polymerase III synthesizes the leading strand continuously.

Lagging Strand Synthesis

• Primase joins RNA nucleotides into a primer.
• DNA polymerase III forms Okazaki fragment 1.
• DNA polymerase III detaches.
• DNA polymerase I replaces RNA with DNA, adding nucleotides.
• DNA ligase seals the bond between new DNA and fragment 1.
• The lagging strand is now complete.

DNA Replication Machine

• Proteins involved in DNA replication form a large complex/ "DNA replication machine."
• Studies suggest DNA polymerase molecules reel in parental DNA and extrude new molecules.
• DNA polymerase reels the parent and extrudes new daughter DNA molecules.

Proofreading and Repair

• DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides.
• Mismatch repair enzymes correct errors in base pairing.
• Nucleotide excision repair involves a nuclease cutting out and replacing damaged DNA stretches.
• Excision repair is triggered by damaged DNA from chemicals or physical agents.
• Enzymes detect and repair damaged DNA, such as thymine dimers.
• A nuclease cuts the damaged DNA, and DNA polymerase fills in the missing nucleotides using the undamaged strand as a template.
• DNA ligase seals the new DNA to the old, completing the strand.
• Errors are low after proofreading but can become permanent mutations and sources of genetic variation.

End of Replication Problem

• DNA polymerase limitations create issues for linear DNA in eukaryotic chromosomes.
• The usual replication machinery can't complete the 5' ends.
• Repeated replication rounds lead to shortening of DNA molecules at the ends.
• Prokaryotes aren't affected, but repeated eukaryotic replication produces shorter DNA molecules with uneven ends.

Telomeres

• Eukaryotic chromosomal DNA molecules have telomeres (special nucleotide sequences) at the ends.
• Telomeres don't prevent DNA shortening but delay erosion of genes near chromosome ends.
• Telomere shortening is linked to aging and cell viability loss, leading to age-related diseases.

Telomerase

• Telomerase catalyzes lengthening of telomeres in germ cells.
• Telomerase counteracts telomere shortening and the end-replication problem.
• Telomerase is expressed by germ cells and early embryonic cells.
• It's not expressed by most somatic human cells.
• Telomerase may be expressed by some stem cells but is highly controlled and often found in cancer cells.

Chromosomes

• Bacterial chromosomes are double-stranded, circular DNA molecules with small amounts of protein.
• In bacteria, DNA is supercoiled and found in the nucleoid.
• Bacteria also have extrachromosomal plasmids, which are duplicated independently.
• Eukaryotic chromosomes are linear DNA molecules associated with a large amount of protein.
• Most eukaryotic DNA is packed in the nucleus. Some DNA is in mitochondria/chloroplasts and duplicates separately.

Chromatin

• DNA in eukaryotic cells is bound to histones, which condense and protect DNA.
• DNA + associated proteins = chromatin.
• Nucleosome: a unit of chromatin (DNA wrapped around a core of eight histones).

Euchromatin and Heterochromatin

• Most chromatin is loosely packed in the nucleus during interphase and condenses before mitosis.
• Loosely packed chromatin is called euchromatin.
• During interphase, regions of chromatin (centromeres and telomeres) are already highly condensed into heterochromatin.
• Histones can have post-translational modifications that affect chromatin organization.

Prior to Mitosis

• When the cell divides, all chromatin highly compacts into visible chromosomes.

Chromosome Painting

• Human chromosomes can be treated with special molecular tags that allow each pair of homologous chromosomes to be seen as a different color.
• This helps distinguish among chromosomes to see how chromosomes are arranged in the interphase nucleus.
• Each chromosome appears to occupy a specific territory during interphase.
• Generally, the two homologues of a pair are not located together.