17.2 Chromosome Organization & DNA Replication

Chromosome Organization & DNA Replication

Quick Answer: Eukaryotic DNA is wound around histone octamers into nucleosomes, condensed into chromosomes, and replicated semiconservatively from origins. Because DNA polymerase synthesizes only 5′→3′, one strand is made continuously (leading) and the other discontinuously (lagging, Okazaki fragments). Per the PA-CAT Bulletin of Information, rev. 20240815, replication of genetic material is tested within the Genetics content area.

Chromatin Packaging Hierarchy

The basic repeat unit of chromatin is the nucleosome: ~147 bp of DNA wrapped ~1.65 turns around an octamer of histones — two copies each of H2A, H2B, H3, H4. The linker histone H1 binds the DNA entering and exiting the nucleosome, stabilizing the 30-nm fiber. Higher-order looping onto a protein scaffold further condenses chromatin into the visible chromosomes of metaphase, achieving a ~10,000-fold compaction of the linear DNA molecule.

Histones are rich in lysine and arginine, giving them a positive charge that neutralizes the negatively charged DNA backbone. Histone tails undergo post-translational modifications — acetylation (generally opens chromatin, activates transcription, via histone acetyltransferases HATs), methylation (context-dependent), phosphorylation, and ubiquitination — collectively called the histone code. These modifications are heritable through cell division as epigenetic marks and are central to the PA-CAT's Molecular Properties of Genes grouping.

• Euchromatin: loosely packed, transcriptionally active, replicates early in S phase.
• Heterochromatin: tightly packed, largely transcriptionally silent (e.g., centromeres, telomeres, Barr body inactivated X). Replicates late in S phase.

A replicated metaphase chromosome consists of two sister chromatids joined at the centromere (primary constriction); the kinetochore assembles on the centromere to anchor spindle microtubules. Chromosome arms are designated p (short, petit) and q (long).

Ploidy and Chromosome Number

Humans are diploid (2n = 46): 22 pairs of autosomes plus one pair of sex chromosomes (XX or XY). Haploid (n = 23) gametes result from meiosis. Homologous chromosomes carry the same genes at the same loci but may carry different alleles; one member of each pair is maternal, one paternal. Ploidy (n, 2n, 3n, etc.) describes the number of complete chromosome sets; aneuploidy is a deviation from a whole-number set (e.g., trisomy 21).

Semiconservative Replication

The Meselson–Stahl experiment (1958) labeled parental DNA with heavy ¹⁵N, grew cells in light ¹⁴N, and observed a single hybrid-density band after one round and a 50:50 mix of hybrid and light bands after two — demonstrating that replication is semiconservative: each daughter double helix retains one parental strand paired with one new strand. Conservative and dispersive models were ruled out.

Initiation

• Origins of replication: eukaryotic chromosomes have many (human ~30,000–50,000), each licensed in G1 by loading the MCM helicase; prokaryotes typically have a single origin (oriC). Multiple origins let the large human genome replicate in ~8 hours.
• Helicase (DnaB in prokaryotes; MCM in eukaryotes) unwinds the double helix in the 5′→3′ direction on the lagging-strand template, creating the replication fork.
• Topoisomerase (DNA gyrase in prokaryotes; Topo I and II in eukaryotes) relieves supercoiling ahead of the fork by cutting and resealing DNA.
• Single-strand binding proteins (SSBPs / RPA) keep the separated strands apart and protect them from nuclease attack.
• Primase lays down a short RNA primer (5–10 nt in prokaryotes, ~10 in eukaryotes) — required because DNA polymerase cannot initiate synthesis de novo, only extend an existing 3′-OH.

Elongation and Fork Asymmetry

DNA polymerase III (prokaryote) or Pol δ/ε (eukaryote) adds nucleotides 5′→3′ and proofreads with 3′→5′ exonuclease activity, removing misincorporated bases immediately (editing function). Error rates after proofreading are ~10⁻⁷. DNA polymerase I (prokaryote) removes RNA primers with its 5′→3′ exonuclease and fills the gap; in eukaryotes, RNase H and FEN1 remove primers and Pol δ fills. DNA ligase seals the remaining nick with ATP (or NAD⁺ in prokaryotes), forming the final phosphodiester bond.

The sliding clamp (β clamp in prokaryotes; PCNA in eukaryotes) tethers polymerase to DNA, dramatically increasing processivity. The clamp-loader complex opens and places the clamp at primer-template junctions.

Mismatch and Post-Replication Repair

Despite proofreading, some errors remain. Mismatch repair (MMR) recognizes mismatches on the newly synthesized strand (identified by transient undermethylation in prokaryotes; nicks in eukaryotes), excises a segment, and resynthesizes. Mutations in MMR genes (e.g., MLH1, MSH2) cause Lynch syndrome (HNPCC), a cancer-predisposition syndrome the PA-CAT may pair with the Medical Genetics grouping.

Telomeres and the End-Replication Problem

Because the lagging strand's final primer cannot be replaced at the very chromosome end, each somatic division loses ~50–100 bp. Human telomeres are 5′-TTAGGG-3′ repeats (thousands of copies) bound by shelterin proteins, forming a protective T-loop that prevents end-to-end fusions. Telomerase — a reverse transcriptase carrying its own RNA template (TERC) — extends telomeric repeats, solving the end-replication problem. It is active in germline cells, stem cells, and ~90% of cancers but inactive in most somatic cells, so telomere shortening acts as a mitotic clock linked to replicative senescence (Hayflick limit). Mutations in telomerase components cause dyskeratosis congenita.

PA-CAT Application

Expect items testing enzyme roles (which enzyme unwinds? which joins fragments? which synthesizes 5′→3′?), the leading vs. lagging distinction, the consequence of semiconservative replication after one vs. two rounds in the Meselson–Stahl density-shift experiment, and the relationship between telomerase activity, senescence, and cancer. Remember: primase makes RNA, ligase makes the final phosphodiester bond, and only DNA polymerase synthesizes new DNA strands.