13. Eight Challenges of Human Genome Replication (967)

Every 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 give eight reasons why replicating the human genome to that level of accuracy is such a monumental task.

Challenge 1: The genome is very large.

The human genome consists of more than three billion nucleotides. Human cells hold two genomes--one from each parent. Both must be replicated. The challenge, then, is making a nearly exact copy of six billion nucleotides in about eight hours.

Six billion of anything is hard to grasp. Let me convert to something more human scale. What would six billion nucleotides long look like in book form? Consider a popular textbook: Campbell’s Biology. It has about 1,250 pages. If the book was filled with the letters A, G, C, and T, it would take about 1,400 of them to hold the human genome. Stacked up, the tower would be over 200 feet high.

 Challenge 2: The genome is tightly packaged.

Replicating six billion or so nucleotides would be difficult enough if the DNA existed in its naked double helical form. But it's not. It's spooled all along its length around proteins called histones in structures called nucleosomes.

The second problem, then, is that the genomic DNA must be unpackaged and repackaged in real time as replication speeds along at roughly 50 nucleotides per second.

Challenge 3: Epigenetic markers must be preserved

Epigenetic markers are informative chemical tags attached to DNA and to the histone proteins of nucleosomes. These marks carry meaning and produce important effects. Some define cell identity. Others promote or impede transcription of genes. Others help recruit DNA repair enzymes. Thus, as a replication fork advances, the cell must not only dismantle and then reassemble nucleosomes, it also must re-establish the original pattern of chemical marks on both the histone proteins and the 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. The first is that it's vulnerable to cleavage by nuclease enzymes roaming around the cell. These enzymes cut DNA and RNA and such damage is difficult 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. So, somehow, exposed single-stranded DNA must be protected.

Challenge 5: Genome regions must be replicated only once

The cell copies its genome quickly by replicating many regions simultaneously, or in parallel. In a dividing cell, about 1,500 replications may be active at any one time, generating a total of about 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 would be copied more than once, dramatically destabilizing the genome.

Challenge 6: Replication errors must be corrected

Replication involves synthesizing a new and complementary DNA strand on a single-stranded DNA template. Rare, potentially highly detrimental mistakes occur. Sometimes the wrong base is attached--such as a C instead of an A across from a T. DNA polymerases also sometimes incorporate RNA nucleotides instead of DNA nucleotides into DNA, introducing chemically unstable components that must be identified and removed. The cell needs mechanisms to first identify and then correct errors caused during replication before replication is completed.

Challenge 7: DNA lesions must be repaired

Replication errors aren't the only kind of DNA problems that the cell must address. DNA exists in a chemically active environment and is susceptible to mutation-causing 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 wide range of repair mechanisms capable of identifying and then fixing a wide variety of DNA lesions.

Challenge 8: Telomeres and DNA sequence repeats

Telomeres are DNA-protein structures at the ends of chromosomes--kind of like the protective aglets at the ends of shoelaces. They consist of long stretches of short, repeated DNA sequences and act as protective caps, preventing chromosome ends from being mistaken for broken DNA. These and other repetitive DNA sequence patterns present a challenge for the replication machinery. Because of the repeats, the DNA polymerase enzyme can lose its place, leading to insertions or deletions.

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

Ultimately, replicating the genome is not one task but a coordinated set of solutions 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.