Congratulations—you made it to the end of the book. That was not an easy read. If you step back, the story I’ve told you is almost absurd. Cells are made of molecules. Molecules don’t plan, anticipate, or understand. They just move--rapidly and randomly--colliding with each other millions of times per second. There is no conductor. There is no blueprint being consulted as events unfold. No molecule knows what the cell is trying to accomplish. And yet, within this chaotic, res

An active replication fork has two possible fates: it collides with another fork moving toward it or it reaches the end of a chromosome. We'll spend most of this chapter focused on the latter case. But first let's consider what happens when two replication forks meet head-on. Head-to-head encounters The majority of replication forks will run into others moving toward it When this occurs, the cell detects the situation and attaches a ubiquitin molecule to the CMG helicases. Th

Imagine the genome as a massive highway construction project. Thousands of crews are paving road at the same time, each working on its own short stretch. Each crew represents a replication fork. Under normal circumstances, if one crew encounters a problem--a cracked pipe or a large rock in the path of a line--they handle it locally. They pause briefly, clear the obstruction, and continue paving. Cells do something similar. When a replication fork encounters a lesion, local me

Let's reset the scene. A replication fork has stalled. The polymerase cannot move forward due to an unrepaired lesion that it has reached. If the lesion isn't immediately repaired or bypassed by TLS or re-priming in the first roughly 20-30 minutes after RPA-single-stranded DNA detection, the fork risks collapsing. The cell's solution to this is highly mechanical: it literally pushes the replication fork backward. This maneuver, called fork reversal, moves the lesion away from

BER and NER remove many DNA lesions before they cause trouble. But they don't always get there on time. Sometimes a lesion is still present when a replication fork arrives. Typically, in such cases, the CMG helicase continues moving and opening the DNA, but the polymerase suddenly stops, impeded by the lesion. The replication fork has entered a dangerous state. Unless the cell responds quickly, the carefully organized replication machinery may come apart and the fork may coll

As we saw in the last chapter, when a DNA polymerase stalls at an unrepaired lesion, the replication fork becomes vulnerable. Within minutes, the cell detects the RPA-coated single-stranded DNA, stabilizes the replisome, and protects the exposed DNA—a collection of responses known as fork protection. From there, molecular pathways compete to determine what happens next: repair the lesion immediately via BER or NER, bypass it and perfrom BER or NER later in S phase, or complet

Base Excision Repair (BER) fixes relatively subtle chemical damage to individual bases. Nucleotide excision repair (NER) tackles a different class of problem altogether. It removes bulky, helix-distorting lesions--damage large enough to warp the double helix. These kinds of lesions cause bulges, kinks, or chemical obstructions in the DNA. They can stall DNA polymerases. And very severe lesions can even interfere with the CMG helicase. There are two ways NER can begin. Here w

In addition to DNA polymerase errors, the human genome--even in a perfectly healthy cell--is under intense assault. The replication errors we just discussed are mistakes made while copying DNA. Lesions are different. They're molecular injuries inflicted throughout the cell cycle on otherwise accurately copied DNA. Lesion-causing threats come from within and from without. Chemicals generated by normal cell metabolism react with DNA. Agents from the environment--sunlight, pollu

Surprisingly, the most common type of DNA polymerase error is not the kind of mismatch, insertion, or deletion we covered in the last chapter. It's the insertion of a RNA nucleotide into a growing chain instead of a DNA nucleotide. When that occurs, the bases do pair correctly, but the small chemical difference between an RNA nucleotide and a DNA nucleotide alters the structure of the DNA backbone and makes it prone to breakage. RNA nucleotide incorporations must be fixed. Ho

DNA polymerases are astonishingly accurate--but not perfect. Let me quantify that. The leading and lagging strand DNA polymerases, which are the workhorses of genome replication, initially make a nucleotide addition mistake once every 10,000 to 100,000 nucleotides. That’s impressive, but not nearly accurate enough. That number of mis-incorporations would result in tens to hundreds of thousands of errors in a replicated human genome. But it's known that when a human genome is

As the CMG helicase moves along the double helix, propelling the replisome forward and separating the DNA into leading and lagging strands, it creates a physical, or topological, problem. DNA can't be pulled apart without repercussions. The act of unwinding one region of the double helix necessarily affects the regions ahead of it. This is often referred to as the “winding problem.” Let me explain with a human-scale analogy. Imagine two ropes wound tightly around each other l

As I explained in the last chapter, DNA replication has a directional problem. The molecular machines that build DNA--the DNA polymerases--can add new nucleotides in only one direction: 5′ to 3′. That rule is absolute, dictated by the chemistry of the reaction itself. But the two strands of the DNA double helix run in opposite directions. They're antiparallel. So what happens when the replication fork moves forward? One strand can be copied smoothly and continuously. The othe

Let's start off with two indisputable facts. One about DNA. The other about DNA polymerases. Fact one. Recall that the two DNA strands in a double helix are oriented in an antiparallel manner. They run in opposite directions like the lanes of a two-lane highway. Scientists describe strand orientation as either “5’-to-3’” (five prime to three prime) or “3’-to-5’” (three prime to five prime) based on the orientations of the sugars in the strand's backbone. Fact two: In the same

Inside the nucleus of every dividing cell during S phase, massive multi-protein rings race along the DNA. Two of them--both already introduced--are the subject of this chapter. The first one--the CMG helicase--is long and barrel-shaped and has an internal motor. It leads the replisome's charge. Using its motor, it propels itself and the replisome forward, prying the double helix apart into two single strands that can be copied. The other--the PCNA sliding clamp--is shaped mor

A human cell enters S phase of the cell cycle. Across its genome, tens of thousands of replication origins have been prepared in advance. Each origin has been licensed, or approved for use, based on the presence of two head-to-head MCMs. This is the MCM double hexamer, or MCM-DH. The two MCMs are the inactive precursors of the two DNA helicases that will lead the replication machinery down the DNA, unwinding the double helix so that both strands can be copied. Initially, the

When the genome is replicated, the cell needs to ensure regions aren't copied multiple times. That would be like a novel in which random sentences, parts of sentences, chapters and parts of chapters are randomly repeated one or more times. In a genome, such repetitions cause serious problems. In this chapter, we take a look at the tight regulation involved in making sure that the cell replicates the genome once and only once with no repeated sections. The mechanism is call

If someone had wanted a book in the 15th century, it would have been copied by hand from an existing one. The scribe would have started at page one and gone page by page until the last page was, as they say, “in the books.” A human genome is like a book. So, to copy it, one might imagine the cell would use the same approach: start at one end of each of the 46 chromosomes and progress to the other end. Unfortunately, that wouldn’t work. Human cells must replicate their genom

Having just reviewed all the steps in the cell cycle, we now focus on the fourth step, mitosis, where the duplicated genome is separated into two new cells. I liken this amazing feat of molecular choreography to a Western-style line dance. Let's review mitosis from start to finish before covering each step in more detail. Our starting point will be the completion of interphase--that is, phases G1, S, and G2. The genome was copied in S phase. All the chromosomes in the nucleus

Every day, your body replaces hundreds of billions of cells. Each must first copy over three billion letters of DNA and, once that's done, divide its contents with extraordinary precision. The importance and rate of cell division vary dramatically between different cell types. Some tissues--like bone marrow, the lining of the gut, the skin, and hair follicles--are constantly turning over. Their cells divide continuously to replace onesc lost to wear. Other cell types divide r

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 ea