🧬Molecular Biology Unit 4 – DNA Replication and Repair
DNA replication is a crucial process that creates identical copies of genetic material. It involves unwinding the double helix, separating strands, and synthesizing new complementary strands using enzymes like DNA polymerase.
DNA repair mechanisms maintain genomic integrity by detecting and fixing errors or damage. These processes include mismatch repair, base excision repair, and nucleotide excision repair, which correct various types of DNA damage and mutations.
DNA replication the process of creating two identical copies of DNA from one original DNA molecule
Semi-conservative replication each strand of the original double helix acts as a template for the synthesis of a new complementary strand
Origin of replication specific sequence in the DNA where replication begins
Replication fork the Y-shaped region where the DNA strands are separated and new strands are synthesized
Leading strand the strand of DNA that is synthesized continuously in the 5' to 3' direction
Lagging strand the strand of DNA that is synthesized discontinuously in short fragments called Okazaki fragments
DNA polymerase the enzyme responsible for catalyzing the synthesis of new DNA strands
DNA ligase the enzyme that joins the Okazaki fragments on the lagging strand to create a continuous strand of DNA
DNA repair mechanisms processes that detect and correct errors or damage in DNA to maintain genomic integrity
DNA Structure Recap
DNA is a double helix structure composed of two antiparallel polynucleotide strands
The strands are held together by hydrogen bonds between complementary base pairs adenine (A) with thymine (T) and guanine (G) with cytosine (C)
The sugar-phosphate backbone provides structural support and consists of alternating deoxyribose sugars and phosphate groups
The nitrogenous bases (A, T, G, and C) are attached to the sugar molecules and project inward from the backbone
The directionality of DNA strands is determined by the 5' and 3' ends referring to the carbon atoms of the deoxyribose sugar
The 5' end has a phosphate group attached to the 5' carbon
The 3' end has a hydroxyl group attached to the 3' carbon
The antiparallel nature of DNA means that one strand runs in the 5' to 3' direction while the complementary strand runs in the 3' to 5' direction
Stages of DNA Replication
Initiation begins at the origin of replication where proteins bind and separate the DNA strands forming the replication bubble
Helicase unwinds and separates the DNA strands by breaking the hydrogen bonds between base pairs
Single-stranded binding proteins (SSBs) stabilize the single-stranded DNA and prevent the strands from reannealing
Elongation involves the synthesis of new DNA strands complementary to the template strands
Primase synthesizes short RNA primers complementary to the template strand providing a starting point for DNA synthesis
DNA polymerase III extends the new DNA strand by adding nucleotides complementary to the template strand in the 5' to 3' direction
The leading strand is synthesized continuously
The lagging strand is synthesized discontinuously as Okazaki fragments
DNA polymerase I replaces the RNA primers with DNA nucleotides
DNA ligase joins the Okazaki fragments to create a continuous strand
Termination occurs when the replication forks from opposite directions meet and the newly synthesized strands are separated from the template strands
Topoisomerases help relieve the tension and supercoiling caused by the unwinding of DNA during replication
Enzymes and Proteins Involved
Helicase unwinds and separates the DNA strands by breaking the hydrogen bonds between base pairs
Single-stranded binding proteins (SSBs) bind to and stabilize single-stranded DNA preventing the strands from reannealing
Topoisomerases relieve the tension and supercoiling caused by the unwinding of DNA
Type I topoisomerases create single-strand breaks and pass one strand through the break before resealing it
Type II topoisomerases create double-strand breaks and pass a double-stranded segment through the break before resealing it
Primase synthesizes short RNA primers complementary to the template strand providing a starting point for DNA synthesis
DNA polymerase III the main enzyme responsible for DNA synthesis extending the new DNA strand by adding nucleotides complementary to the template strand
It has a high processivity meaning it can add many nucleotides before dissociating from the template
It also has proofreading activity allowing it to remove incorrectly incorporated nucleotides
DNA polymerase I replaces the RNA primers with DNA nucleotides
DNA ligase seals the nicks between Okazaki fragments creating a continuous strand of DNA
Replication Mechanisms
Semi-conservative replication each strand of the original DNA molecule serves as a template for the synthesis of a new complementary strand
The original strands are separated and each acts as a template
New strands are synthesized complementary to the template strands
The result is two identical DNA molecules each containing one original strand and one newly synthesized strand
Bidirectional replication DNA replication proceeds simultaneously in both directions from the origin of replication
Two replication forks are formed moving in opposite directions
This mechanism allows for faster replication of large DNA molecules
Continuous synthesis on the leading strand the new DNA strand is synthesized continuously in the 5' to 3' direction
DNA polymerase III adds nucleotides to the growing strand without interruption
Discontinuous synthesis on the lagging strand the new DNA strand is synthesized in short fragments (Okazaki fragments) in the 5' to 3' direction
DNA polymerase III synthesizes each Okazaki fragment starting from an RNA primer
The fragments are later joined by DNA ligase to create a continuous strand
Proofreading and error correction DNA polymerases have proofreading activity that allows them to remove incorrectly incorporated nucleotides
The 3' to 5' exonuclease activity of DNA polymerase III can excise mismatched nucleotides
This mechanism helps maintain the accuracy of DNA replication
DNA Repair Processes
Mismatch repair corrects errors that occur during DNA replication when incorrect nucleotides are incorporated
Mismatch repair enzymes recognize and excise the mismatched nucleotide
DNA polymerase fills in the gap with the correct nucleotide and DNA ligase seals the nick
Base excision repair removes damaged or modified bases (oxidized, alkylated, or deaminated) from DNA
Glycosylases recognize and remove the damaged base creating an apurinic/apyrimidinic (AP) site
AP endonuclease cleaves the phosphodiester backbone at the AP site
DNA polymerase fills in the gap and DNA ligase seals the nick
Nucleotide excision repair removes bulky DNA lesions (thymine dimers, pyrimidine dimers) caused by UV light or chemicals
Enzymes recognize the distortion in the DNA helix caused by the lesion
Endonucleases make incisions on both sides of the lesion and remove the damaged segment
DNA polymerase fills in the gap using the undamaged strand as a template and DNA ligase seals the nick
Double-strand break repair fixes breaks in both strands of the DNA molecule caused by ionizing radiation or chemicals
Homologous recombination uses the sister chromatid as a template to repair the break accurately
Non-homologous end joining directly ligates the broken ends but is more error-prone
Common Errors and Mutations
Point mutations changes in a single nucleotide resulting from substitution, insertion, or deletion
Substitution replaces one nucleotide with another (transition or transversion)
Insertion adds one or more extra nucleotides
Deletion removes one or more nucleotides
Frameshift mutations insertions or deletions that alter the reading frame of the genetic code
Frameshift mutations can lead to completely different amino acid sequences or premature stop codons
Chromosomal mutations large-scale changes in the structure or number of chromosomes
Deletions remove a portion of a chromosome
Duplications repeat a portion of a chromosome
Inversions reverse the orientation of a chromosomal segment
Translocations transfer a segment from one chromosome to another
Spontaneous mutations occur naturally due to errors in DNA replication or repair
Tautomeric shifts in DNA bases can lead to mispairing during replication
Depurination loss of a purine base (A or G) creates an AP site that can lead to mutations if not repaired
Induced mutations result from exposure to mutagens (chemicals, radiation, or viruses) that damage DNA or interfere with its replication
Alkylating agents (nitrosamines, mustard gas) add alkyl groups to DNA bases leading to mispairing
Intercalating agents (ethidium bromide, proflavine) insert between base pairs and distort the DNA helix
UV light causes the formation of pyrimidine dimers that can lead to mutations if not repaired
Real-World Applications and Research
DNA sequencing technologies (Sanger sequencing, next-generation sequencing) rely on the principles of DNA replication to determine the nucleotide sequence of DNA
These technologies have revolutionized fields such as genomics, personalized medicine, and evolutionary biology
Polymerase chain reaction (PCR) a technique that amplifies specific DNA sequences using DNA polymerase and primers
PCR has numerous applications in molecular biology, genetic testing, forensic science, and infectious disease diagnosis
CRISPR-Cas9 a powerful gene-editing tool derived from bacterial adaptive immune systems
CRISPR-Cas9 uses guide RNA to target specific DNA sequences and the Cas9 endonuclease to create double-strand breaks
This technology has the potential to correct genetic disorders, create disease-resistant crops, and develop novel therapies
DNA damage and repair in disease DNA damage and defects in repair mechanisms are associated with various diseases
Xeroderma pigmentosum a rare genetic disorder characterized by extreme sensitivity to UV light and increased risk of skin cancer due to defects in nucleotide excision repair
Hereditary nonpolyposis colorectal cancer (Lynch syndrome) caused by mutations in mismatch repair genes leading to increased risk of colorectal and other cancers
Fanconi anemia a genetic disorder caused by mutations in genes involved in DNA repair leading to bone marrow failure and increased risk of leukemia and solid tumors
Cancer and genomic instability the accumulation of mutations and chromosomal abnormalities is a hallmark of cancer
Oncogenes (Ras, Myc) and tumor suppressor genes (p53, BRCA1/2) are often mutated in cancer cells
Defects in DNA repair pathways (mismatch repair, nucleotide excision repair, double-strand break repair) can lead to increased mutation rates and genomic instability in cancer