Genetics: DNA Forms and Replication

• Genetics involves a study of DNA

DNA Forms

• Three forms of DNA spirals exist: B-form, A-form, and Z-form

B-form

• Features a right-handed twist
• Represents the most abundant DNA configuration
• Found as DNA on DNA
• Consists of 10 nucleotides per 360° turn

A-form

• Features a right-handed twist
• Can be either DNA on DNA or DNA on RNA
• Composed of 11 nucleotides per 360° turn
• Known as the dehydrated form

Z-form

• Features a left-handed twist
• Contains 12 bases per 360° turn
• Forms a zig-zag shaped DNA
• Supposedly employed as a transcription signal
• Exists as DNA on DNA

DNA replication

• Replication starts by separating DNA strands at multiple points
• These locations are rich in adenine (A) and thymine (T) bases
• These are called "points of origin or consensus areas."
• Bacterial DNA has one point of origin
• The enzyme that separates DNA initially is DNA-A protein
• The open (unzipped) area becomes a replication bubble
• Single-strand binding proteins (SSB proteins) bind to DNA strands, stabilizing the replication bubble and preventing re-annealing
• SSB proteins also protect individual DNA strands from being cleaved by endonucleases
• Helicase is an enzyme that enters each replication fork site to further unwind the DNA, pushing ahead and separating the DNA downstream
• Helicase requires energy to break hydrogen bonds
• Helicase's movement causes supercoils (tightly twisted zones) downstream
• Supercoils are resolved by topoisomerase

Topoisomerase

• Includes a nuclease and a ligase domain
• The nuclease domain of topoisomerase I cuts one DNA strand in supercoiled regions, untwisting supercoils
• The breaks are then glued back together by the ligase domain
• Topoisomerase I has one nuclease domain and cuts one strand
• Topoisomerase II has two nuclease domains, cutting both strands when DNA molecules tangle during replication
• Quinilones are antibiotics that deactivate the ligase domains of topoisomerases, stimulating nuclease
• Orinolones act on bacterial topoisomerase
• This results in DNA getting cleaved but not re-annealed, leading to bacterial death
• Anticancer drugs like etoposides and teniposides target human topoisomerase in cancer cells

DNA Polymerase

• An enzyme called primase (DNA-dependent RNA polymerase) initiates the replication process
• Primase comes to the replication bubble and synthesizes RNA complementary to the DNA segment, forming a primer
• The primer provides the necessary 3' end of the nucleotide chain, which DNA-dependent DNA polymerase uses to start building the DNA chain
• DNA polymerase reads the template DNA from 3' to 5' direction
• DNA polymerase synthesizes a new strand 5' to 3'
• He DNA polymerase will proofread the new strand from 3' to 5'
• DNA polymerase uses its exonuclease domain to remove any mismatched nucleotides
• The polymerase reads the last nucleotide on the template again, and then inserts the correct nucleotide
• After proofreading to ensure the the sequence is correct, it moves to the next nucleotide in the template DNA strand

Leading and Lagging Strand

• The new DNA strand synthesized off the primer in the direction of the replication fork is called the leading strand, and is synthesized without interruption
• A new DNA strand that made off the primer away from the replication fork is called a lagging strand
• The lagging strand is synthesized in shorter fragments off new primers as the replication fork advances due to DNA's spiral
• Primase travels along the template strand with other proteins in a complex called the primosome in order to create these primers
• Okazaki fragments refers to the shorter DNA pieces on a lagging strand from one primer to another
• DNA polymerase I does everything that DNA polymerase III enzyme, but it also possesses another exonuclease domain that can eliminate RNA bonds from 5', thus correcting fragmented DNA on the lagging strand replacing it with the correct DNA

DNA Synthesis Inhibitors

• A class of drugs, pseudo-nucleotides or pseudo-nucleosides, have modified deoxyribose lacking an -OH group on carbon #3
• DNA polymerase III or DNA polymerase I cannot recognize these altered sugars and adds these modified nucleotides to the synthesized DNA strand due to the lack of an -OH group on the 3' end
• These drugs include antivirals and anticancer agents
  • Didinozine - antiviral, anti-HIV (analog of inosine)
  • Vidarabine - antiviral (adenosine analog)
  • Acyclovir - antiviral (guanine analog)
  • Cytarabine - anticancer (cytosine analog)
  • Zidavudine - anti-HIV (thymidine analog)

Mitotic Spindle Inhibitors

• Some attack the spindle, while others stabilize it
• There are two medications that attack the mitotic spindle of dividing cells: vincristine and vinblastine
  • These destroy tubulin, the building block of the mitotic spindle
  • Vinblastine is more aggressive than vincristine and causes bone marrow ablation
• Paclitaxel and docetaxel hyper-stabilize the mitotic spindle, preventing shrinkage and chromatid separation

Eukaryotic Polymerases

• The previously mentioned polymerases are only found in prokaryotic cells
• Eukaryotic cells utilize a variation of polymerases:
  • α-pol: serves as a primer and creates short DNA strands
  • β-pol: involved in DNA repair, correcting mismatched bases
  • γ-pol: replicates mitochondrial DNA
  • δ-pol: elongates the leading strand and forms Okazaki fragments
  • ε-pol: also plays a role in DNA repair

Telomeres

• During DNA replication, when the lagging strand reaches the template's end, it is unable to replicate to the very end given the lack of a 3' end, which can result in a genetic information loss
• Telomerase adds telomere, which extends the end of chromosomes
• It also has its own RNA template complementary to the telomere (TTAGGG) sequence
• The last 2 bases of the telomerase pair with the last 2 bases on the DNA strand
• The enzyme initiates reverse transcription to elongate the DNA
• Telomerase leaves once the DNA is long enough before primase can start a new DNA strand
• The template of the removed primer is removed once the primer is used to template the start of a chain
• Telomerase elongates the DNA strand, ensuring that the overhang is not the DNA
• Large amounts of telomerase are found in stem cells, and cancer cells upregulate telomerase activity to allow for constant replication utilizing cancer cells.