13. Eight Challenges of Genome Replication (1,089)

Each time a cell divides, the cell copies its entire genome—billions of nucleotides—with extraordinary accuracy. After proofreading and repair, there will be only one error every 10–100 million nucleotides!

In this chapter, I review eight reasons why replicating the human genome to that level of accuracy is such a monumental task.

The cell overcomes these difficulties, of course. As evidence, consider that on the developmental path from embryo to adult, human cells carry out trillions of successful cell divisions.

Challenge 1: The genome is very large.

The human genome consists of more than three billion nucleotides. Human cells hold two versions of the genome--one from each parent--and both must be replicated.

The challenge, then, is making a nearly exact copy of six billion nucleotides in about six to ten hours. Six billion of anything is hard to grasp. So let me convert that number to a more human scale.

What would a genetic code six billion nucleotides long look like in book form? Consider a popular biology textbook: Campbell’s Biology. It has about 1,250 pages.

If, instead of biology chapters, the book was filled with the letters A, G, C, and T in the same font, it would take about 1,400 of such textbooks to hold the human genome. Stacked up, the tower would be over 200 feet high.

The cell has an enormous amount of DNA to copy.

Challenge 2: The genome is packaged.

Replicating a genome that size would be difficult enough if the DNA existed in its naked form. But that’s not the case.

The second problem is that genomic DNA must be dynamically unpackaged and repackaged as replication proceeds-- at on the order of about 50 nucleotides per second at each replication fork.

To do this, the cell locally opens chromatin rather than unwinding the higher-order fiber structures completely.

Proteins then move or remove nucleosomes ahead of the replication machinery, allowing the DNA to be accessed and copied, after which the chromatin is reassembled.

So not only is there a vast amount of DNA--but DNA that is tightly packaged and must be unpacked and repacked in real time.

Challenge 3: Epigenetic markers on histones must be preserved

As if removing and replacing nucleosomes wasn't enough, their epigenetic markers must also be preserved.

Cells chemically mark histone tails in specific ways that carry meaning and produce important effects. Some define cell identity. Others promote or impede transcription of specific genes. Some mark regions as tightly packaged heterochromatin or loosely packaged euchromatin. Others help recruit DNA repair enzymes.

Scientists refer to a "histone code" when discussing these modifications, and deciphering this code is a very active area of research.

In addition to histones, DNA itself is chemically marked in meaningful ways.

As the replication fork advances, the cell must not only dismantle and swiftly reassemble nucleosomes, it also must largely re-establish the original pattern of chemical marks on both histones and DNA.

Challenge 4:  Single-stranded DNA must be protected

Replicating the genome requires continuously separating the two strands of the double helix. Single-stranded DNA, however, is susceptible to two problems: enzymatic cleavage and the formation of secondary structures.

Single-stranded DNA is more vulnerable to cleavage by nucleases--enzymes that cut DNA and RNA--and such damage is difficult for the cell to repair.

In addition, because of complementary base pairing, single stranded DNA can fold back on itself and form secondary structures--regions of unwanted double stranded-ness that can impede the replication machinery.

The cell therefore needs mechanisms to protect exposed single-stranded DNA during replication.

Challenge 5: The genome must be replicated only once

The cell copies its genome quickly by replicating many regions simultaneously.

In a dividing cell, about 1,500 replications may be active at once, generating 10,000 to 20,000 replication forks during S phase.

Given this parallelism, the cell must somehow keep track of which regions have been replicated and which have not. Without that control, segments of the genome could be copied more than once, quickly destabilizing the genome.

Challenge 6: Replication errors must be corrected

Replication involves synthesizing a complementary strand on a single-stranded template, and rare mistakes occur.

Sometimes the wrong base is attached--such as a C across from a T instead of an A. DNA polymerases can also incorporate ribonucleotides into DNA, introducing chemically unstable components that must be identified and removed.

The cell needs mechanisms to identify and correct such errors accurately before replication is completed.

Challenge 7: DNA lesions must be repaired

DNA exists in a chemically active environment and is susceptible to damage from both internal and external sources.

Sometimes the base on a nucleotide becomes chemically altered. Occasionally, the two DNA strands become chemically attached to each other. Other times proteins errantly chemically attach to DNA.

The cell must therefore maintain a range of repair mechanisms capable of addressing different types of DNA lesions.

Challenge 8: Telomeres and DNA sequence repeats

Telomeres are specialized DNA-protein structures at the ends of chromosomes. They consist of short, repeated DNA sequences--specifically the sequence 5'-TTAGGG-3' typically repeated over 1,000 times.

Telomeres act as protective caps, preventing chromosome ends from being mistaken for broken DNA and triggering inappropriate repair responses.

These and other highly repetitive sequences are challenging for the replication machinery. Because the same short sequence is repeated, the polymerase can lose its place, leading to small insertions or deletions. Also, telomeric DNA and other repeats can form stable secondary structures that impede replication.

Telomeres also present a unique problem due to their position at chromosome ends. For reasons that will become clear later, the replication machinery can't fully copy one end of the DNA strand, leading to the gradual loss of telomeric DNA with each cell division--a phenomenon known as the end-replication problem.

This problem must be addressed in cells that divide repeatedly over long periods of time.

Replicating the genome is not a single task but a coordinated solution to many concurrent problems—copying a vast amount of DNA, preserving its organization, protecting fragile intermediates, and correcting inevitable errors and lesions.

What appears to be a smooth process is really the result of many overlapping systems working together with amazing precision.

In the chapters that follow, we’ll examine how the cell meets these challenges--step by step--at the level of individual molecules.

A Human-Scale Genome: If the A, G, C and Ts of the human genome were written on every page of a 1,250 page textbook, it would take a stack of 1,400 books to capture it.

Meaningful chemical markings: These include epigenetic factors attached to histone tails and direct methylation of DNA nucleotides.