Molecular Biology of the Cell

• MBOC, Fifth Edition (2008)
• Authors: Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter
• Excellent 30th anniversary retrospective of how MBOC originally came together in the late 1970s, how its first edition was published in 1983, and the impact its various editions have had on education and research over the decades. By Norberto Serpente in Nature, 2013. Located on Bruce Albert’s website.

Chapter 1: Cells and Genomes

• p. 20 - defintion of homologs: All related genes that result from duplication
  • Duplicate genes that are conserved in two branches after speciation are called orthologs
  • Duplicate genes within a single genome are paralogs
  • Orthologs and paralogs are subsets of the larger containing set of homologs
• Dehydration Synthesis (aka Condensation Synthesis) vs. Hydrolysis. See Alberts 5e Fig. 2-64 and 2-65 on p. 85, Alberts 5e Fig. 2-19 on p. 57, and JH handwritten notes (8/22/2020)

Chapter 2: Cell Chemistry and Biosynthesis

10 Steps of Glycolysis

• Glycolysis converts glucose to pyruvate in 10 steps.
• For more details, see JH handwritten notes August 7-13, 2020, Alberts p. 120-121 (Panel 2-8), and this page.

Chapter 3: Proteins

• See p. 127-129 to see tables and panels of categories of polar vs. nonpolar sidechain/amino acids, acidic vs. basic sidechains, etc.

Chapter 4: Chromosome Structure and Genomes

• Avg. size of a human gene is ~27,000 bp including both introns and exons. However, avg size of genes is only 1300 bp when all the introns are thrown out. (p. 206)
• See other interesting stats on human genes in Table 4-1 on p. 206.
• Shared synteny aka conserved synteny = not only are DNA sequences / genes conserved, but the exact order of these genes are conserved across species (p. 207).
• Three specialized DNA sequences are special on the chromosome: (1) origin of replication; prokaryotes have 1 origin per chromosome while eukaryotes have multiple ori’s; (2) centromere; (3) telomere.

Chapter 5: DNA Replication and Repair

• DNA structure on Panel 2-6 Alberts p. 116-17 and Figure 4-4 and 4-5 on p. 198-199. See also JH handwritten notes August 25-29, 2020.
• Mutation rates in DNA are surprisingly low across multiple genera. It is about 1 mutation in 10⁹ in both E. coli and C. elegans aka 1 base pair change per 1 billion bp. In addition, more indirect methods in humans using fibrinopeptides, fibrinogen, and fibrin formation during blood clotting have also produced a similar 1 in a billion figure (p. 264-265).
• See Table 5-1 on p. 271 to see how the 3 different error correcting mechanisms of 5’→ 3’ polymerization, 3’→ 5’ exonuclease proofreading, and strand-directed mismatch repair together combine to provide a 1 error per 10⁹ bp during DNA replication.
• DNA Polymerase I was discovered by Arthur Kornberg in 1956 ( p. 266)
• Okazaki fragments discovered in mid- to late-1960s in Japan. This along with other evidence disproved earlier hypothesis that DNA polymerases could act in both 5’→ 3’ and in the 3’→ 5’ direction.

Proofreading mechanisms during and post replication

1. Delay between initial base pair hydrogen bonding and permanent phosophodiester covalent bonding. First, a free nucleotide base pairs with the template DNA strand via hydrogen bonding. Obviously, the correct nucleotide is energetically more favorable. However, DNA polymerase is like a right hand whose fingers must tighten for the next step: covalently creating the phosphodiester bond on the backbone. It’s hard to do that with the wrong nucleotide. So even if the wrong nucleotide slips in for step 1a, it’s hard to “permanently” lock it in with the “finger-closing step”. (p. 269 and Fig 5-4b)
2. Exonucleolytic proof-reading that excise nucleotides dNMP (aka 2’-deoxyribose Nucleotide Mono Phosphates) one at a time from the new daughter strand. The exonuclease is actually a separate catalytic site on DNA polymerase. DNA Poly “absolutely requires a previously formed base-paired 3’-OH end of the primer strand.” If the wrong dNMP is at the end of the daughter strand, the exonuclease is engaged and excised in the opposite 3’→ 5’ direction from the Poly’s regular growing 5’→ 3’ direction. (See Fig. 5-8 and 5-9 on p. 270.)
3. Strand-Directed Mismatch Repair System which is a post-replication complex that catches errors missed in the initial multi-enzyme replication machine. This system finds mismatched base-pairs between a template and daughter strand. How does it know which is the template and which is the new strand?
   • In prokaryotes like E.coli, by looking for unmethlyated A’s. In bacteria, new strands eventually get all the A’s in the sequence GATC methylated. Fresh new daughter strands have not gotten their A’s methylated so that is the strand that is corrected. (p. 276-277)
   • Eukaryotes rarely (never?) have their A’s methylated. Fresh, new daughter strands are identified because they have frequent ss nicks where ligase has not performed it’s function.

Why is DNA growth only in the 5’→ 3’ direction?

For answer, see Fig. 5-10 p. 271

Multi-enzyme Replication Complex per Alberts Fig. 5-19

• See p. 276 Fig 5-19…
• DNA helicase to unwind DNA template strands at the replication fork. See the example of dnaB helicase in E. coli and this 2020 intellectual history of heilcases.
• Primase to put down RNA primers. Used in leading strand once but used over and over again in the lagging strand.
• DNA Polymerase that both adds new nucleotides and has an exonuclease catalyic site to remove mismatches
• Sliding clamp and clamp loader to keep DNA Poly machinery attached to template strand
• Single-Stranded DNA Binding Protein (aka SSB) used only on the lagging strand
• Ligase used on lagging strand to stitch together the various Okazaki fragments and to replace RNA primers with proper DNA segments.
• Also two other items:
  • topo I and topo II to relieve DNA helix torsion ahead of the replication fork.
  • strand-directed mismatch repair, keyed to unmethylated A nucleotides in GATC sequences.
• A more technical anatomy of the replisome can be found on the Wikipedia entry for E. coli Pol III See more recent developments this 2018 paper on bacterial replisomes

Major enzymes involved in DNA Replication per Lander lecture

• Helicase to make the initial cut to start the replication fork
• Primase to add short RNA primers as a starting point for DNA Polymerase
• DNA Polymerase and associated Exonuclease catalytic sites
• Ligase to join together short Okazaki fragments on the lagging strand
• Topoisomerase I and II that moves ahead of the replication fork and relieving the coil tension created by unwinding the double helix. Topo I creates a single strand nick while Topo II is a gate-folded enzyme that moves strands past each other with a controlled cut and rejoin of one strand. See also p. 278-280.

Right-handed structure of Polymerase

• Per this link, all DNA polymerases have three domains:
  1. The “thumb” binds to the DNA substrate
  2. The “fingers” recognize specific nucleotides and performs the base-pair hydrogen bonding
  3. The “palm” contains the catalytic sites. (I think this means the “P” catalytic site for the phosphodiester covalent bond extending the DNA backbone and the “E” catalytic site for proofreading and exonuclease for incorrect nucleotides that have been accidentally attached.)

Good links for Replisome

• Wikipedia article on the replisome
• Clamp loader ATPases and the evolution of DNA replication machinery (2012) by Kelch, Makino, O’Donnell, & John Kuriyan. Picture suggests that there are three Pol III cores attached to the replisome. One is always attached to the leading strand while the second and third polymerase enzymes switch off on alternate Okazaki fragments. This view is different than the animation shown in Berry’s 2010 replication video which only shows a total of two Pol III cores, one for each strand.
• Michael O’Donnell’s website at Rockefeller University
  • Publications
  • Replisome mechanics: insights into a twin DNA polymerase machine (2007)
  • DNA replication in prokaryotes
  • DNA replication in eukaryotes

Different types of DNA Polymerase

Prokaryotic DNA Polymerase

• Pol I - first polymerase discovered by Arthur Kornberg in 1956. “Pol I accounts for 95% of the polymerase activity in E. coli” but not primarily in replication. Removes RNA primers on the lagging strand and replaces them with DNA to connect Okazaki fragments. According to Wikipedia, Pol I has 4 functions:
  1. Most important replication function: forward 5’→ 3’ replacement of RNA with proper DNA daughter sequences. This all takes place on the lagging strand and connects Okazaki fragments, waiting only for ligase to seal everything up to finish the job.
  2. Proofreading in the reverse 3’→ 5’ direction using exonuclease site.
  3. Forward exonuclease 5’→ 3’ to mediate nick translation during DNA repair.
  4. Very low usage forward 5’→ 3’ RNA-dependent polymerase activity.
• Pol II was discovered in 1970 by Thomas Kornberg and there is 5x more abundant in E. coli cells as compared to Pol III.
  • The activity of Pol II is still under debate but it seems to be a backup molecule for replication when Pol III stalls out.
  • Pol II seems to preferentially work on the lagging strand.
  • Pol II important to the E. coli SOS induction cellular repair system.
  • Part of Famiy B like eukaryotic polymerases like Pol B, Pol alpha(α), Pol delta (δ), Pol epsilon (ε).
• Pol III
  • Although lowest in apparent concentration and activity, Pol III is by far the most important polymerase used in bacterial DNA replication.
  • Because of the action of it’s associated sliding camp, this polymerase works incredibly, fast, moving through nucleotides at a 1000x rate compared to other polymerases.
  • Like Pol II, discovered in 1970 by Thomas Kornberg.
  • Member of Family C. Moves in the usual 5’→ 3’ direction for replication and moves backwards in the 3’→ 5’ direction for exonuclease action.
  • Has 3 components: (1) the pol III core, (2) the beta sliding clamp processivity factor, and (3) the sliding clamp loader. The clamp loader is responsible for both attaching the clamp to the DNA strand and removing the clamp. A sliding camp molecule can only be attached to either a clamp loader complex or a polymerase molecule, but not both at the same time.
• Pol IV –involved in E. coli SOS response cellular repair system
• Pol V –involved in E. coli SOS response cellular repair system
• Family D

Eukaryotic DNA Polymerase

• Polymerases alpha (α), delta (δ), and epsilon (ε) - part of Family B
  • Polymerase delta (δ) is probably the eukaryotic equivalent of Pol III in bacteria because polymerase delta (δ) also has an associated sliding clamp called PCNA. Per the DNA Clamp Wikipedia article, PCNA is highly conserved across the animal, fungi, and plant kingdoms.
• Polymerase beta (β) used in DNA repair
• Polymerase lambda (λ), sigma (σ), and mu (μ) and TdT
• Polymerases eta (η), iota (ι), and kappa (κ)
• Polymerases Rev1 and zeta (ζ)
• Polymerases gamma (γ) is used in mitochondria. theta (θ), and nu (ν)
• Reverse Transcriptase
• Telomerase

7 families of DNA polymerase

• Family A - Pol I and has 2 exonuclease domains (goes in both directions 5’→ 3’ and 3’→ 5’)
• Family B - Pol II for proofreading
• Family C - Prokaryotic only, e.g. Pol III Standard workhorse for primary E. coli transcription
• Family D (Archaea)
• Family X - Eukaryotic only
• Family Y
• Family RT - viruses, retroviruses, and Eukaryotes, e.g., telomerase

Chapter 7: Control of Gene Expression

• 9/11/2020