DNA Replication and Repair Processes

• DNA replication and repair are essential processes for maintaining genetic information

DNA Replication

• Variations in DNA sequences lead to differences between individuals, even within the same family.
• Base pairing facilitates DNA replication

Base Pairing and DNA Replication

• Each of the two complementary strands of DNA serves as a template for synthesizing a new strand

Semi-Conservative Replication

• DNA replication is semi-conservative, each daughter DNA helix contains one old (conserved) and one new strand

DNA Synthesis at Replication Origins

• Initiator proteins bind to specific DNA sequences at replication origins (ORI) to begin DNA synthesis
• Initiator proteins unzip short regions of the double helix at normal temperatures

Nature of Replication

• There are three models for DNA replication

Models for DNA Replication

• Semiconservative: Each parent strand acts as a template for synthesizing a new daughter strand
  • Each daughter helix has one parental and one newly synthesized strand
• Dispersive: Parent and newly synthesized DNA are interspersed in a mosaic pattern
• Conservative: The parent molecule remains intact after being copied
  • Yields both the original parent double helix and a completely new double helix

Meselson-Stahl Experiment

• Bacteria were grown on media containing a heavy isotope to label DNA
• Cells were broken, and DNA was loaded into an ultracentrifuge tube containing a cesium chloride salt solution
• Tubes were centrifuged at high speeds to form a gradient
• Formation of a gradient causes, DNA to migrate to a region where its density matches that of the surrounding salt
  • Heavy and light DNA molecules collect in different positions within the tube
• Heavy isotope N15 was denser, resulting in a gradient closer to the bottom of the tube
• The same hypothesis was tested with E. coli; the resulting generation (from adding a heavy isotope to light) was a hybrid containing both isotopes
  • The conservative model was ruled out
  • Heat was added in order to break the hydrogen bonds

Replication Forks

• DNA synthesis occurs at Y-shaped junctions called replication forks, which move away from each other as they "unzip" the double strand in opposite directions
• Replication is bidirectional and moves rapidly at approximately 100 nucleotide pairs per second

DNA Polymerase

• DNA polymerase synthesizes DNA using a parent strand as a template
• DNA synthesis occurs in the 5' to 3' direction; every nucleotide pair is added to the 3' end of the new strand

Nucleosides and Nucleic Acids

• Nucleoside consists of a base plus a sugar
• Nucleotide includes a nucleoside with one or more phosphate groups
• Nucleic acid refers to long chains of nucleotides that carry genetic information

Polymerization Reaction

• DNA polymerase catalyzes the addition of nucleotides to the growing 3' end
• Polymerization reaction involves the formation of a phosphodiester bond between the 3' end and the 5' phosphate group
• The reaction is guided by DNA polymerase and requires proper base pairing
• Proper base pairing allows the 5' triphosphate to react with the 3'-OH on the growing strand, while polymerase moves in the 5' to 3' direction, ensuring correct nucleotide addition

Replication Fork Asymmetry

• One strand runs 5' to 3' (template strand) while the other runs 3' to 5' (new strand)

DNA Polymerase Direction

• DNA polymerase 3 reads the template in the 3' to 5' direction but writes in the 5' to 3' direction

Leading and Lagging Strands

• The leading strand is a newly synthesized DNA strand that is continuously synthesized in the 5' to 3' direction
• The leading strand template is the template strand that guides the synthesis of the leading strand, running in the 3' to 5' direction
• All newly synthesized strands are either leading or lagging, depending on their direction relative to the replication fork

DNA Polymerase: Self-Correction

• Proofreading occurs when DNA polymerase makes a mistake and adds the wrong nucleotide
  • Proofreading corrects the error
  • It occurs simultaneously with DNA synthesis

DNA Polymerase Structure

• DNA polymerase 3 contains separate sites for DNA synthesis and proofreading
  • P (Polymerizing)= polymerizing
  • E (Editing) = editing/proofreading, occurs from 5' to 3'
• Polymerase adds the incorrect nucleotide, the new strand temporarily unpairs and moves to the E site to be corrected

RNA Primers

• Short RNAs act as primers

How Replication Starts

• Replication begins with short RNA primers, synthesized by primase, providing a starting point for polymerase to add nucleotides
• RNA primers are complementary to single-stranded DNA templates
• Once DNA synthesis proceeds, the RNA is replaced by DNA
• Primase synthesizes RNA using DNA as a template

Synthesis of Lagging Strand

• RNA primers (approximately 10 nucleotides long) are extended by DNA polymerase 3 to form Okazaki fragments
• RNA primers removed by nucleases that degrade the RNA strand
• The gaps left are filled by DNA polymerase 1
• DNA ligase joins the Okazaki fragments by forming phosphodiester bonds
  • The ligation process requires ATP to provide the energy for the reaction
• DNA ligase joins Okazaki fragments on the lagging strand during DNA synthesis via ATP hydrolysis, resulting in adenine monophosphate

Telomeres

• Telomeres are at the ends of eukaryotic chromosomes

Eukaryotic Chromosome Replication

• DNA replication risks losing chromosome ends
  • Replication starts at origins and continues to chromosome ends
  • The leading strand is fully synthesized
  • The lagging strand cannot be completed due to the removal of the final RNA primer

Telomerase Function

• Telomerase prevents chromosome end shrinkage during cell division

Preventing Shortening

• Telomerase extends the template strand beyond copied DNA
• The extended template allows primase to lay down RNA primers
• Telomerase uses RNA as a template to synthesize telomere DNA helping to maintain genetic information
• Telomere length varies by cell type and with age
• Telomeres are repetitive DNA sequences at the ends of chromosomes
  • They protect chromosomes from damage and prevent them from fusing with other chromosomes

Telomere Variation

• Different cell types have varying telomere lengths due to differences in their rate of division and the purposes they serve in the body
  • Stem cells and germ cells tend to maintain longer telomeres because they need to divide extensively and remain functional over a lifetime
  • Somatic cells have shorter telomeres because they divide fewer times, resulting in telomere loss with each division
• Telomeres are sometimes compared to the plastic tips at the ends of shoelaces (aglets), as they help maintain chromosome integrity

DNA Repair

• DNA damage occurs continually in cells

Common Chemical Reactions

• Depurination and deamination are the most frequent chemical reactions known to create serious DNA damage
• Depurination is the process that removes guanine or adenine from DNA, leading to possible mutations
• Deamination refers to the conversion of cytosine to uracil, an abnormal base in DNA
  • Other bases can also undergo deamination

Impact on DNA

• Both deamination and depurination can alter the genetic code without breaking the phosphodiester backbone
• Consequences of these modifications can lead to errors during DNA replication and repair, resulting in mutations
• Reactions are common sources of DNA damage but don't disrupt the structural integrity of the DNA helix
• Ultraviolet radiation in sunlight can cause the formation of thymine dimers, where covalent linkages are formed between adjacent pyrimidine bases, resulting in dimer formation

DNA Repair Mechanisms

• Cells possess a variety of mechanisms for repairing DNA

Steps for Repairing DNA

• Excision: A segment of the damaged strand is excised
• Resynthesis: DNA polymerase fills in the missing nucleotide in the top strand using the bottom strand as a template
• Ligation: DNA ligase seals the nick

Mismatch Repair System

• A DNA mismatch repair system removes replication errors that escape proofreading
• Mismatch repair is dedicated to correcting errors and corrects 99% of replication errors
• Mispaired nucleotides are called a mismatch
• If left uncorrected, mismatches can cause permanent mutations, often prevalent in cancer predisposition

Double-Strand Breaks

• Double-strand DNA breaks require a different strategy for repair
• Cells can repair double-strand breaks in two ways

Double-Strand Repair Methods

• Nonhomologous End Joining: Rapid sticking of DNA broken fragments back, however, it can result in a loss of nucleotides at the site of repair
• Homologous Recombination: Flawless repair of double-strand breaks with no loss of genetic information
  • A recombination-specific nuclease chews back the 5' ends of broken strands
  • Specialized enzymes, one of the broken 5' ends, invade unbroken DNA and looks for regions of compliment

Consequences of Failure to Repair DNA

• Failure to repair DNA damage can have severe consequences for a cell or an organism
• For example, a single nucleotide change in the beta-globin gene results in a specific amino acid change
  • Glutamic acid gets replaced by valine

DNA replication and repair are essential processes for maintaining genetic information. Variations in DNA sequences lead to differences between individuals, even within the same family. DNA replication is semi-conservative, each daughter DNA helix contains one old and one new strand.